Aryl N-cyanoguanidines and methods related thereto

ABSTRACT

Compounds, compositions and methods treating arthritic disorders such as osteoarthritis or rheumatoid arthritis, and for treating other diseases associated with altered mitochondrial function, such as cancer, psoriasis, stroke, Alzheimer&#39;s Disease and diabetes. The compounds of this invention have the following structure (I):  
                 
 
     including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein R 1-5  are as defined herein. The methods of this invention are directed to administering to a warm-blooded animal in need thereof an effective amount of a compound of structure (I), typically in the form of a pharmaceutical composition.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of U.S. patent applicationSer. No. 10/085,119, filed Feb. 27, 2002, now allowed; and claims thebenefit of U.S. Provisional Application No. 60/272,368 filed Feb. 27,2001, which applications are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

[0002] The present invention relates generally to compounds and methodsfor treating arthritis and related disorders, and for treating diseasesassociated with altered mitochondrial function and, more particularly,to aryl N-cyanoguanidine compounds and derivatives thereof.

BACKGROUND OF THE INVENTION

[0003] Numerous chronic debilitating diseases of the skeletal system invertebrates, including arthritis and related arthritic disorders,feature degradation of specialized avascular cartilaginous tissue knownas articular cartilage that contains dedicated cartilage-producingcells, the articular chondrocytes. Unlike other chondrocytes such asepiphyseal growth plate chondrocytes present at the ends of developinglong bones (e.g., endochondral or costochondral chondrocytes), articularchondrocytes reside in and maintain joint cartilage having novasculature. Thus lacking a blood supply as an oxygen source, articularchondrocytes are believed to generate metabolic energy, for examplebioenergetic ATP production, predominantly by anaerobic (e.g.,glycolytic) respiration, and not from aerobic mitochondrial oxidativephosphorylation (Stefanovich-Racic et al., J. Cell Physiol. 159:274-80,1994). Because even under aerobic conditions, articular chondrocytes mayconsume little oxygen and thus appear to differ from a wide variety ofvertebrate cell types (Stefanoviceh-Racic et al., 1994), mitochondrialroles in arthritic disorders have been largely ignored.

[0004] The musculoskeletal system efficiently delivers useful mechanicalenergy and load support in vertebrates such as mammals, reptiles, birdsand fish, but is also capable of synthesizing, processing and organizingcomplex macromolecules to fashion tissues and organs specialized toperform specific mechanical functions. The joints are an importantsubset of the specialized structures of the musculoskeletal system, andmany distinct types of joints exist in the body. Freely moving joints(e.g., ankle, elbow, hip, knee, shoulder, and joints of the fingers,toes and wrist) are known as diarthrodial or synovial joints. Incontrast, the intervertebral joints of the spine are not diarthrodialjoints as they are fibrous and do not move freely, although they doprovide the flexibility required by the spine. The articulating boneends in the diarthrodial joint are lined with a thin layer of hydratedsoft tissue known as articular cartilage. Fourth, the joint isstabilized by, and its range of motion controlled by, ligaments andtendons that may be inside or outside the joint capsule.

[0005] The surface linings of diarthrodial joints, i.e., the synoviumand articular cartilage layers, form the joint cavity that contains thesynovial fluid. Thus, in vertebrate skeletal joints, the synovial fluid,articular cartilage, and the supporting bone form a smooth, nearlyfrictionless bearing system. While diarthrodial joints are subjected toan enormous and varied range of load conditions, the cartilage surfacesundergo little wear and tear (e.g., structural degradation) under normalcircumstances. Indeed, most human joints are capable of functioningeffectively under very high loads and stresses and at very low operatingspeeds. These performance characteristics demand efficient lubricationprocesses to minimize friction and wear of cartilage in the joint.Severe breakdown of the joint cartilage by biochemical and/orbiomechanical processes leads to arthritis, which is therefore generallydefined as a failure of the vertebrate weight bearing system.

[0006] Articular chondrocytes synthesize and deposit the components of,and reside in, a three-dimensional cartilaginous extracellular matrixcomprised largely of two major classes of macromolecules, collagen andproteoglycans. Articular chondrocytes thus mediate the synthesis,assembly, degradation and turnover of the macromolecules which comprisethe cartilage extracellular matrix (ECM or simply “matrix”).Mechanochemical properties of this matrix contribute significantly tothe biomechanical function of cartilage in vivo.

[0007] The structural integrity of articular cartilage is the foundationof optimal functioning of the skeletal joints, such as those found inthe vertebrate hip, shoulders, elbows, hocks and stifles. Impairedskeletal joint function dramatically reduces an individual subject'smobility, such as that involved in rising from a sitting position or inclimbing and descending stairs. As noted above, in order to maintain thestructural and functional integrity of articular cartilage, articularchondrocytes constantly synthesize collagen and proteoglycans, the majorcomponents of the articular cartilage; chondrocytes also secrete thefriction-reducing synovial fluid. This constant elaboration by articularchondrocytes of cartilage ECM macromolecules and synovial fluid providesthe articular cartilage with a repair mechanism for most mechanical wearthat may be caused by friction between the bone ends. However, suchsteady biosynthesis of cartilage components generates a constant demandfor the precursors, or building blocks, of these macromolecules andsynovial fluid components. Lack of these precursors will lead to defectsin the structure and function of the skeletal joints. This deficiencyoccurs often when activity levels are very high, or when cartilagetissue is traumatized.

[0008] The menisci of the knee, and other similar structures such as thedisc of the temporomandibular joint and the labrum of the shoulder, arespecialized fibrocartilagenous structures that are vital for normaljoint function. They are known to assist articular cartilage indistributing loads across the joint, to aid ligaments and tendons instabilizing joints and to play a major role in shock absorption, and mayfurther assist in lubricating the joint. Damage to these structures canlead to impaired joint function and to articular cartilage degeneration.Surgical removal of these fibrocartilagenous structures, for example,following apparently irreparable cartilage tears, can result in earlyonset of osteoarthritis. The menisci, disc and labrum are hydratedfibrocartilage structures composed primarily of type II collagen, withsmaller amounts of other collagens and proteoglycans (including aggrecanand the smaller, non-aggregating proteoglycans) also present. Thesefibrocartilaginous structures contain a sparse population of residentcells that, like the articular chondrocytes of cartilage, areresponsible for the synthesis and maintenance of this extracellularmatrix.

[0009] Diarthrodial joints enable common bodily motions including limbmovements associated with motor (e.g., ambulatory) functions and otheractivities of daily life. Failure of the joint surfaces (i.e., articularcartilage) means a failure of these biomechanical bearings to providetheir central functions, such as ambulatory and other bodily motion,delivery of mechanical energy and load support.

[0010] In biomedical terms, failure of diarthrodial joints leads toarthritic disorders, the most common forms being osteoarthritis ordegenerative joint disease, or chondrocalcinosis. Other forms ofarthritic disorders include but are not limited to rheumatoid arthritis,juvenile rheumatoid arthritis, ankylosing spondylitis, Reiter'ssyndrome, psoriatic arthritis, lupus erythematosous, gout, infectiousarthritides and chondrocalcinosis (see, e.g., Gilliland et al.,“Disorders of the joints and connective tissue,” Section 14, Harrison'sPrinciples of Internal Medicine, Eighth Ed., Thorn et al., eds.McGraw-Hill, New York, N.Y., 1977, pp. 2048-80) and, in a veterninarycontext, dysplasias such as canine hip dysplasia. Arthritic disorderscan also include, or may result from, physical trauma (for example,acute physical injury that damages joint tissue, or repetitive motionsyndrome) or dietary conditions (e.g., ricketts or other dietarydeficiency diseases) that result in joint injury.

[0011] By far, the most prevalent arthritic disorders are rheumatoidarthritis (RA) and osteoarthritis (OA). RA, thought to be an autoimmunedisorder, results in part from inflammation of the synovial membrane. Inhumans, peak onset of this disorder occurs in adults over 30 years ofage (typically in their thirties and forties) and afflicts women threetimes more often than men. In extreme cases, chronic inflammation erodesand distorts the joint surfaces and connective tissue, resulting insevere articular deformity and constant pain. Moreover, RA often leadsto OA, further compounding the destruction of the joint. The most commonarthritic disorder, OA, is characterized by degenerative changes in thesurface of the articular cartilage. Alterations in the physicochemicalstructure of the cartilage make it less resistant to compressive andtensile forces. Finally, complete erosion occurs, leaving thesubchondral bone exposed and susceptible to wear. Joints of the kneesand hands are most often affected, as also may be one or more of thespine, hips, ankles and shoulders. In both RA and OA, degeneration ofthe weight bearing joints such as the hips and knees can be especiallydebilitating and often requires surgery to relieve pain, and to increasemobility.

[0012] No means currently exist for halting or reversing thedegenerative changes brought about by RA, OA and related arthriticdisorders. At the same time, approximately 37 million Americans seeksymptomatic relief in the form of prescription drugs. In such casesnonsteroidal, anti-inflammatory drugs (NSAIDS) are most oftenprescribed. While these compounds often alleviate or palliate thearthritic symptoms, they frequently have undesirable side effects, forexample, nausea and gastrointestinal ulceration. Other compoundscommonly prescribed for the treatment of arthritic disorders are thecorticosteroids, such as triamcinolone, prednisolone and hydrocortisone.These drugs also have undesirable side effects, particularly where longterm use may be required, and so may be contraindicated in manypatients. In addition to difficulties in determining effective dosages,a number of adverse reactions have been reported during intra-articulartreatment with these and other steroids. As a result, the use ofcorticosteroid treatments in the management of arthritic disorders iscurrently being reassessed.

[0013] As an alternative to symptomatic and palliative measures fortreating OA and RA as described above, mechanical repair of arthriticjoints, when feasible, involves orthopedic surgery to replace wornjoints with an artificial prosthesis, or with a biological graft. Withmore than thirty million Americans suffering from these disablingdiseases, such surgery poses enormous medical and economic challengesand is not without its own risks and contraindications.

[0014] As noted above, osteoarthritis, also known as degenerative jointdisease, is one of the most common types of arthritis. It ischaracterized by the breakdown of the cartilage within a joint, causingpainful rubbing of one bone of the joint against another bone andleading to a loss of movement within the affected joint. Osteoarthritiscan range from very mild to very severe, and most commonly affectsmiddle-aged and older people. In particular, OA often affects hands andweight-bearing joints such as the knees, hips, feet and back. Althoughage is a leading risk factor, at present the etiology and pathogenesisof this condition remain largely unknown. Many environmental factors andother independent conditions have been associated with an increased riskfor having or developing osteoarthritis, including obesity, previousinjury and/or menisectomy (e.g., sports-related injuries or otheraccidental injury), occupation-related activities that involve repeatedknee bending, smoking, sex hormones, gynecological disorders and othermetabolic factors. Chondrocalcinosis is another form of degenerativejoint disease related to osteoarthritis, in which abnormal calcificationoccurs in the articular cartilage.

[0015] From the foregoing, it is clear that none of the currentpharmacological therapies corrects the underlying biochemical defect inarthritic disorders such as RA and OA. Neither do any of these currentlyavailable treatments improve all of the physiological abnormalities inarthritic disorders such as abnormal articular chondrocyte activity,cartilage degradation, articular erosion and severe joint deformity. Inaddition, treatment failures are common with these agents, such thatmulti-drug therapy is frequently necessary.

[0016] Clearly there is a need for improved therapeutics that aretargeted to correct biochemical and/or metabolic defects responsible forarthritis. The present invention provides compositions and methods thatare useful for treating an arthritic disorder and for treating otherdiseases, and offers other related advantages.

[0017] According to non-limiting theory, and as disclosed in theco-pending application having U.S. Ser. No. 09/661,848, which isincorporated by reference, some or all arthritic disorders as providedherein may represent examples of diseases associated with alteredmitochondrial function.

[0018] By way of background, mitochondria are the main energy source incells of higher organisms, and these organelles provide direct andindirect biochemical regulation of a wide array of cellular respiratory,oxidative and metabolic processes (for a review, see Ernster and Schatz,J. Cell Biol. 91:227s-255s, 1981.). These include electron transportchain (ETC) activity, which drives oxidative phosphorylation to producemetabolic energy in the form of adenosine triphosphate (ATP), and whichalso underlies a central mitochondrial role in intracellular calciumhomeostasis. In addition to their role in metabolic processes,mitochondria are also involved in the genetically programmed cellsuicide sequence known as “apoptosis” (Green and Reed, Science281:1309-12, 1998; Susin et al., Biochim. et Biophys. Acta 1366:151-65,1998).

[0019] Defective mitochondrial activity, including but not limited tofailure at any step of the elaborate multi-complex mitochondrialassembly, known as the electron transport chain (ETC), may result in (i)decreases in ATP production, (ii) increases in the generation of highlyreactive free radicals (e.g., superoxide, peroxynitrite and hydroxylradicals, and hydrogen peroxide), (iii) disturbances in intracellularcalcium homeostasis and (iv) the release of factors (such as such ascytochrome c and “apoptosis inducing factor”) that initiate or stimulatethe apoptosis cascade. Because of these biochemical changes,mitochondrial dysfunction has the potential to cause widespread damageto cells and tissues.

[0020] A number of diseases and disorders are thought to be caused by orbe associated with alterations in mitochondrial metabolism and/orinappropriate induction or suppression of mitochondria-relatedfunctions, such as those leading to apoptosis. These include, by way ofexample and not limitation, chronic neurodegenerative disorders such asAlzheimer's disease (AD) and Parkinson's disease (PD); auto-immunediseases; diabetes mellitus, including Type I and Type II; mitochondriaassociated diseases, including but not limited to congenital musculardystrophy with mitochondrial structural abnormalities, fatal infantilemyopathy with severe mtDNA depletion and benign “later-onset” myopathywith moderate reduction in mtDNA, MELAS (mitochondrial encephalopathy,lactic acidosis, and stroke) and MIDD (mitochondrial diabetes anddeafness); MERFF (myoclonic epilepsy ragged red fiber syndrome);arthritis; NARP (Neuropathy; Ataxia; Retinitis Pigmentosa); MNGIE(Myopathy and external ophthalmoplegia; Neuropathy; Gastro-Intestinal;Encephalopathy), LHON (Leber's; Hereditary; Optic; Neuropathy),Kearns-Sayre disease; Pearson's Syndrome; PEO (Progressive ExternalOphthalmoplegia); Wolfram syndrome DIDMOAD (Diabetes Insipidus, DiabetesMellitus, Optic Atrophy, Deafness); Leigh's Syndrome; dystonia;schizophrenia; and hyperproliferative disorders, such as cancer, tumorsand psoriasis. The extensive list of additional diseases associated withaltered mitochondrial function continues to expand as aberrantmitochondrial or mitonuclear activities are implicated in particulardisease processes.

[0021] According to generally accepted theories of mitochondrialfunction, proper ETC respiratory activity requires maintenance of anelectrochemical potential (ΔΨm) in the inner mitochondrial membrane by acoupled chemiosmotic mechanism. Conditions that dissipate or collapsethis membrane potential, including but not limited to failure at anystep of the ETC, may thus prevent ATP biosynthesis and hinder or haltthe production of a vital biochemical energy source. Altered ordefective mitochondrial activity may also result in a catastrophicmitochondrial collapse that has been termed “mitochondrial permeabilitytransition” (MPT). In addition, mitochondrial proteins such ascytochrome c and “apoptosis inducing factor” may dissociate or bereleased from mitochondria due to MPT (or the action of mitochondrialproteins such as Bax), and may induce proteases known as caspases and/orstimulate other events in apoptosis (Murphy, Drug Dev. Res. 46:18-25,1999).

[0022] Defective mitochondrial activity may alternatively oradditionally result in the generation of highly reactive free radicalsthat have the potential of damaging cells and tissues. These freeradicals may include reactive oxygen species (ROS) such as superoxide,peroxynitrite and hydroxyl radicals, and potentially other reactivespecies that may be toxic to cells. For example, oxygen free radicalinduced lipid peroxidation is a well established pathogenetic mechanismin central nervous system (CNS) injury such as that found in a number ofdegenerative diseases, and in ischemia (i.e., stroke). (Mitochondrialparticipation in the apoptotic cascade is believed to also be a keyevent in the pathogenesis of neuronal death.)

[0023] There are, moreover, at least two deleterious consequences ofexposure to reactive free radicals arising from mitochondrialdysfunction that adversely impact the mitochondria themselves. First,free radical mediated damage may inactivate one or more of the myriadproteins of the ETC. Second, free radical mediated damage may result incatastrophic mitochondrial collapse that has been termed “transitionpermeability”. According to generally accepted theories of mitochondrialfunction, proper ETC respiratory activity requires maintenance of anelectrochemical potential in the inner mitochondrial membrane by acoupled chemiosmotic mechanism. Free radical oxidative activity maydissipate this membrane potential, thereby preventing ATP biosynthesisand/or triggering mitochondrial events in the apoptotic cascade.Therefore, by modulating these and other effects of free radicaloxidation on mitochondrial structure and function, the present inventionprovides compositions and methods for protecting mitochondria that arenot provided by the mere determination of free radical induced lipidperoxidation.

[0024] For example, rapid mitochondrial permeability transition likelyentails changes in the inner mitochondrial transmembrane proteinadenylate translocase that results in the formation of a “pore.” Whetherthis pore is a distinct conduit or simply a widespread leakiness in themembrane is unresolved. In any event, because permeability transition ispotentiated by free radical exposure, it may be more likely to occur inthe mitochondria of cells from patients having mitochondria associateddiseases that are chronically exposed to such reactive free radicals.

[0025] Altered (e.g., increased or decreased in a statisticallysignificant manner relative to an appropriate control, such as adisease-free individual) mitochondrial function characteristic of themitochondria associated diseases may also be related to loss ofmitochondrial membrane electrochemical potential by mechanisms otherthan free radical oxidation, and such transition permeability may resultfrom direct or indirect effects of mitochondrial genes, gene products orrelated downstream mediator molecules and/or extramitochondrial genes,gene products or related downstream mediators, or from other known orunknown causes. Loss of mitochondrial potential therefore may be acritical event in the progression of mitochondria associated ordegenerative diseases.

[0026] Diabetes mellitus is a common, degenerative disease affecting 5to 10 percent of the population in developed countries. The propensityfor developing diabetes mellitus is reportedly maternally inherited,suggesting a mitochondrial genetic involvement. (Alcolado, J. C. andAlcolado, R., Br. Med. J. 302:1178-80, 1991; Reny, S. L., InternationalJ. Epidem. 23:886-90, 1994.) Diabetes is a heterogenous disorder with astrong genetic component; monozygotic twins are highly concordant andthere is a high incidence of the disease among first-degree relatives ofaffected individuals.

[0027] At the cellular level, the degenerative phenotype that may becharacteristic of late onset diabetes mellitus includes indicators ofaltered mitochondrial respiratory function, for example impaired insulinsecretion, decreased ATP synthesis and increased levels of reactiveoxygen species. Studies have shown that diabetes mellitus may bepreceded by or associated with certain related disorders. For example,it is estimated that forty million individuals in the U.S. suffer fromlate onset impaired glucose tolerance (IGT). IGT patients fail torespond to glucose with increased insulin secretion. A small percentageof IGT individuals (5-10%) progress to insulin deficient non-insulindependent diabetes (NIDDM) each year. Some of these individuals furtherprogress to insulin dependent diabetes mellitus (IDDM). These forms ofdiabetes mellitus, NIDDM and IDDM, are associated with decreased releaseof insulin by pancreatic beta cells and/or a decreased end-organresponse to insulin. Other symptoms of diabetes mellitus and conditionsthat precede or are associated with diabetes mellitus include obesity,vascular pathologies, peripheral and sensory neuropathies, blindness anddeafness.

[0028] Due to the strong genetic component of diabetes mellitus, thenuclear genome has been the main focus of the search for causativegenetic mutations. However, despite intense effort, nuclear genes thatsegregate with diabetes mellitus are known only for rare mutations inthe insulin gene, the insulin receptor gene, the adenosine deaminasegene and the glucokinase gene. Accordingly, mitochondrial defects, whichmay include but need not be limited to defects related to the discretenon-nuclear mitochondrial genome that resides in mitochondrial DNA, maycontribute significantly to the pathogenesis of diabetes mellitus(Anderson, Drug Dev. Res. 46:67-79, 1999).

[0029] Parkinson's disease (PD) is a progressive, chronic,mitochondria-associated neurodegenerative disorder characterized by theloss and/or atrophy of dopamine-containing neurons in the pars compactaof the substantia nigra of the brain. Like Alzheimer's Disease (AD), PDalso afflicts the elderly. It is characterized by bradykinesia (slowmovement), rigidity and a resting tremor. Although L-Dopa treatmentreduces tremors in most patients for a while, ultimately the tremorsbecome more and more uncontrollable, making it difficult or impossiblefor patients to even feed themselves or meet their own basic hygieneneeds.

[0030] It has been shown that the neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces parkinsonismin animals and man at least in part through its effects on mitochondria.MPTP is converted to its active metabolite, MPP+, in dopamine neurons;it then becomes concentrated in the mitochondria. The MPP+ thenselectively inhibits the mitochondrial enzyme NADH:ubiquinoneoxidoreductase (“Complex I”), leading to the increased production offree radicals, reduced production of adenosine triphosphate, andultimately, the death of affected dopamine neurons.

[0031] Mitochondrial Complex I is composed of 40-50 subunits; most areencoded by the nuclear genome and seven by the mitochondrial genome.Since parkinsonism may be induced by exposure to mitochondrial toxinsthat affect Complex I activity, it appears likely that defects inComplex I proteins may contribute to the pathogenesis of PD by causing asimilar biochemical deficiency in Complex I activity. Indeed, defects inmitochondrial Complex I activity have been reported in the blood andbrain of PD patients (Parker et al., Am. J. Neurol. 26:719-23, 1989;Swerdlow and Parker, Drug Dev. Res. 46:44-50, 1999).

[0032] Similar theories have been advanced for analogous relationshipsbetween mitochondrial defects and other neurological diseases, includingAlzheimer's disease, Leber's hereditary optic neuropathy, schizophrenia,“mitochondrial encephalopathy, lactic acidosis, and stroke” (MELAS), and“myoclonic epilepsy ragged red fiber syndrome” (MERRF).

[0033] For example, Alzheimer's disease (AD) is a chronic, progressiveneurodegenerative disorder that is characterized by loss and/or atrophyof neurons in discrete regions of the brain, and that is accompanied byextracellular deposits of β-amyloid and the intracellular accumulationof neurofibrillary tangles. It is a uniquely human disease, affectingover 13 million people worldwide. It is also a uniquely tragic disease.Many individuals who have lived normal, productive lives are slowlystricken with AD as they grow older, and the disease gradually robs themof their memory and other mental faculties. Eventually, they cease torecognize family and loved ones, and they often require continuous careuntil their eventual death.

[0034] There is evidence that defects in oxidative phosphorylationwithin the mitochondria are at least a partial cause of sporadic AD. Theenzyme cytochrome c oxidase (COX), which makes up part of themitochondrial electron transport chain (ETC), is present in normalamounts in AD patients; however, the catalytic activity of this enzymein AD patients and in the brains of AD patients at autopsy has beenfound to be abnormally low. This suggests that the COX in AD patients isdefective, leading to decreased catalytic activity that in some fashioncauses or contributes to the symptoms that are characteristic of AD.

[0035] One hallmark pathology of AD is the death of selected neuronalpopulations in discrete regions of the brain. Cell death in AD ispresumed to be apoptotic because signs of programmed cell death (PCD)are seen and indicators of active gliosis and necrosis are not found(Smale et al., Exp. Neurolog. 133:225-30, 1995; Cotman et al., Molec.Neurobiol. 10:19-45, 1995.) The consequences of cell death in AD,neuronal and synaptic loss, are closely associated with the clinicaldiagnosis of AD and are highly correlated with the degree of dementia inAD (DeKosky et al., Ann. Neurol. 27(5):467-64, 1990).

[0036] Mitochondrial dysfunction is thought to be critical in thecascade of events leading to apoptosis in various cell types (Kroemer etal., FASEB J. 9:1277-87, 1995), and may be a cause of apoptotic celldeath in neurons of the AD brain. Altered mitochondrial physiology maybe among the earliest events in PCD (Zamzami et al., J. Exp. Med.182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72, 1995) andelevated reactive oxygen species (ROS) levels that result from suchaltered mitochondrial function may initiate the apoptotic cascade(Ausserer et al., Mol. Cell. Biol. 14:5032-42, 1994). In several celltypes, including neurons, reduction in the mitochondrial membranepotential (ΔΨm) precedes the nuclear DNA degradation that accompaniesapoptosis. In cell-free systems, mitochondrial, but not nuclear,enriched fractions are capable of inducing nuclear apoptosis (Newmeyeret al., Cell 70:353-64, 1994). Perturbation of mitochondrial respiratoryactivity leading to altered cellular metabolic states, such as elevatedintracellular ROS, may occur in mitochondria associated diseases and mayfurther induce pathogenetic events via apoptotic mechanisms.

[0037] Oxidatively stressed mitochondria may release a pre-formedsoluble factor that can induce chromosomal condensation, an eventpreceding apoptosis (Marchetti et al., Cancer Res. 56:2033-38, 1996). Inaddition, members of the Bcl-2 family of anti-apoptosis gene productsare located within the outer mitochondrial membrane (Monaghan et al., J.Histochem. Cytochem. 40:1819-25, 1992) and these proteins appear toprotect membranes from oxidative stress (Korsmeyer et al, Biochim.Biophys. Act. 1271:63, 1995). Localization of Bcl-2 to this membraneappears to be indispensable for modulation of apoptosis (Nguyen et al.,J. Biol. Chem. 269:16521-24, 1994). Thus, changes in mitochondrialphysiology may be important mediators of apoptosis. To the extent thatapoptotic cell death is a prominent feature of neuronal loss in AD,mitochondrial dysfunction may be critical to the progression of thisdisease and may also be a contributing factor in other mitochondriaassociated diseases.

[0038] Focal defects in energy metabolism in the mitochondria, withaccompanying increases in oxidative stress, may be associated with AD.It is well-established that energy metabolism is impaired in AD brain(Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J.Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275, 1989; Balazset al., Neurochem. Res. 19:1131-37, 1994; Mecocci et al., Ann. Neurol.36:747-51, 1994; Gsell et al., J. Neurochem. 64:1216-23, 1995). Forexample, regionally specific deficits in energy metabolism in AD brainshave been reported in a number of positron emission tomography studies(Kuhl, et al., J. Cereb. Blood Flow Metab. 7:S406, 1987; Grady, et al.,J. Clin. Exp. Neuropsychol. 10:576-96, 1988; Haxby et al., Arch. Neurol.47:753-60, 1990; Azari et al., J. Cereb. Blood Flow Metab. 13:438-47,1993). Metabolic defects in the temporoparietal neocortex of AD patientsapparently presage cognitive decline by several years. Skin fibroblastsfrom AD patients display decreased glucose utilization and increasedoxidation of glucose, leading to the formation of glycosylation endproducts (Yan et al., Proc. Nat. Acad. Sci. U.S.A. 91:7787-91, 1994).Cortical tissue from postmortem AD brain shows decreased activity of themitochondrial enzymes pyruvate dehydrogenase (Sheu et al., Ann. Neurol.17:444-49, 1985) and α-ketoglutarate dehydrogenase (Mastrogiacomo etal., J. Neurochem. 6:2007-14, 1994), which are both key enzymes inenergy metabolism. Functional magnetic resonance spectroscopy studieshave shown increased levels of inorganic phosphate relative tophosphocreatine in AD brain, suggesting an accumulation of precursorsthat arises from decreased ATP production by mitochondria (Pettegrew etal., Neurobiol. of Aging 15:117-32, 1994; Pettigrew et al., Neurobiol.of Aging 16:973-75, 1995). In addition, the levels of pyruvate, but notof glucose or lactate, are reported to be increased in the cerebrospinalfluid of AD patients, consistent with defects in cerebral mitochondrialelectron transport chain (ETC) activity (Parnetti et al., Neurosci.Lett. 199:231-33, 1995).

[0039] Signs of oxidative injury are prominent features of AD pathologyand, as noted above, reactive oxygen species (ROS) are criticalmediators of neuronal degeneration. Indeed, studies at autopsy show thatmarkers of protein, DNA and lipid peroxidation are increased in AD brain(Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J.Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275-82, 1989;Balazs et al., Arch. Neurol. 4:864, 1994; Mecocci et al., Ann. Neurol.36:747-51, 1994; Smith et al., Proc. Nat. Acad. Sci. USA. 88:10540-43,1991). In hippocampal tissue from AD but not from controls, carbonylformation indicative of protein oxidation is increased in neuronalcytoplasm, and nuclei of neurons and glia (Smith et al., Nature382:120-21, 1996). Neurofibrillary tangles also appear to be prominentsites of protein oxidation (Schweers et al., Proc. Nat. Acad. Sci. USA.92:8463, 1995; Blass et al., Arch. Neurol. 4:864, 1990). Under stressedand non-stressed conditions incubation of cortical tissue from AD brainstaken at autopsy demonstrate increased free radical production relativeto non-AD controls. In addition, the activities of critical antioxidantenzymes, particularly catalase, are reduced in AD (Gsell et al., J.Neurochem. 64:1216-23, 1995), suggesting that the AD brain is vulnerableto increased ROS production. Thus, oxidative stress may contributesignificantly to the pathology of mitochondria associated diseases suchas AD, where mitochondrial dysfunction and/or elevated ROS may bepresent.

[0040] Increasing evidence points to the fundamental role ofmitochondrial dysfunction in chronic neurodegenerative diseases (Beal,Biochim. Biophys. Acta 1366:211-23, 1998), and recent studies implicatemitochondria for regulating the events that lead to necrotic andapoptotic cell death (Susin et al., Biochim. Biophys. Acta 1366:151-68,1998). Stressed (by, e.g., free radicals, high intracellular calcium,loss of ATP, among others) mitochondria may release pre-formed solublefactors that can initiate apoptosis through an interaction withapoptosomes (Marchetti et al., Cancer Res. 56:2033-38, 1996; Li et al.,Cell 91:479-89, 1997). Release of preformed soluble factors by stressedmitochondria, like cytochrome c, may occur as a consequence of a numberof events. In any event, it is thought that the magnitude of stress(ROS, intracellular calcium levels, etc.) influences the changes inmitochondrial physiology that ultimately determine whether cell deathoccurs via a necrotic or apoptotic pathway. To the extent that apoptoticcell death is a prominent feature of degenerative diseases,mitochondrial dysfunction may be a critical factor in diseaseprogression.

[0041] In contrast to chronic neurodegenerative diseases, neuronal deathfollowing stroke occurs in an acute manner. A vast amount of literaturenow documents the importance of mitochondrial function in neuronal deathfollowing ischemia/reperfusion injury that accompanies stroke, cardiacarrest and traumatic injury to the brain. Experimental support continuesto accumulate for a central role of defective energy metabolism,alteration in mitochondrial function leading to increased oxygen radicalproduction and impaired intracellular calcium homeostasis, and activemitochondrial participation in the apoptotic cascade in the pathogenesisof acute neurodegeneration.

[0042] A stroke occurs when a region of the brain loses perfusion andneurons die acutely or in a delayed manner as a result of this suddenischemic event. Upon cessation of the blood supply to the brain, tissueATP concentration drops to negligible levels within minutes. At the coreof the infarct, lack of mitochondrial ATP production causes loss ofionic homeostasis, leading to osmotic cell lysis and necrotic death. Anumber of secondary changes can also contribute to cell death followingthe drop in mitochondrial ATP. Cell death in acute neuronal injuryradiates from the center of an infarct where neurons die primarily bynecrosis to the penumbra where neurons undergo apoptosis to theperiphery where the tissue is still undamaged (Martin et al., Brain Res.Bull. 46:281-309, 1998).

[0043] Much of the injury to neurons in the penumbra is caused byexcitotoxicity induced by glutamate released during cell lysis at theinfarct focus, especially when exacerbated by bioenergetic failure ofthe mitochondria from oxygen deprivation (MacManus and Linnik, J.Cerebral Blood Flow Metab. 17:815-32, 1997). The initial trigger inexcitotoxicity is the massive influx of Ca²⁺ primarily through the NMDAreceptors, resulting in increased uptake of Ca²⁺ into the mitochondria(reviewed by Dykens, “Free radicals and mitochondrial dysfunction inexcitotoxicity and neurodegenerative diseases” in Cell Death andDiseases of the Nervous System, V. E. Koliatos and R. R. Ratan, eds.,Humana Press, New Jersey, pp. 45-68, 1999). The Ca²⁺ overload collapsesthe mitochondrial membrane potential (ΔΨm) and induces increasedproduction of reactive oxygen species (Dykens, J Neurochem 63:584-91,1994; Dykens, “Mitochondrial radical production and mechanisms ofoxidative excitotoxicity” in The Oxygen Paradox, K. J. A. Davies, and F.Ursini, eds., Cleup Press, U. of Padova, pages 453-67, 1995). If severeenough, ΔΨ_(m) collapse and mitochondrial Ca²⁺ sequestration can induceopening of a pore in the inner mitochondrial membrane through a processcalled mitochondrial permeability transition (MPT), indirectly releasingcytochrome c and other proteins that initiate apoptosis (Bernardi etal., J. Biol. Chem. 267:2934-39, 1994; Zoratti and Szabo, Biochim.Biophys. Acta 1241:139-76, 1995; Ellerby et al., J Neurosci 17:6165-78,1997). Consistent with these observations, glutamate-inducedexcitotoxicity can be inhibited by preventing mitochondrial Ca²⁺ uptakeor blocking MPT (Budd and Nichols, J Neurochem 66:403-11, 1996; Whiteand Reynolds, J Neurosci 16:5688-97, 1996; Li et al., Brain Res.753:133-40, 1997).

[0044] Whereas mitochondria-mediated apoptosis may be critical indegenerative diseases, it is thought that disorders such as cancerinvolve the unregulated and undesirable growth (hyperproliferation) ofcells that have somehow escaped a mechanism that normally triggersapoptosis in such undesirable cells. Enhanced expression of theanti-apoptotic protein, Bcl-2 and its homologues is involved in thepathogenesis of numerous human cancers. Bcl-2 acts by inhibitingprogrammed cell death and overexpression of Bcl-2, and the relatedprotein Bcl-xL, block mitochondrial release of cytochrome c frommitochondria and the activation of caspase 3 (Yang et al, Science275:1129-32, 1997; Kluck et al., Science 275:1132-36, 1997; Kharbanda etal., Proc. Natl. Acad. Sci. U.S.A. 94:6939-42, 1997). In contrast,overexpression of Bcl-2 and Bcl-xL protect against the mitochondrialdysfunction preceding nuclear apoptosis that is induced bychemotherapeutic agents. In addition, acquired multi-drug resistance tocytotoxic drugs is associated with inhibition cytochrome c release thatis dependent on overexpression of Bcl-xL (Kojima et al., J. Biol. Chem.273:16647-50, 1998). Because mitochondria have been implicated inapoptosis, it is expected that agents that interact with mitochondrialcomponents will effect a cell's capacity to undergo apoptosis. Thus,agents that induce or promote apoptosis in hyperproliferative cells areexpected to be useful in treating hyperproliferative disorders anddiseases such as cancer.

[0045] Thus, alteration of mitochondrial function has great potentialfor a broad-based therapeutic strategy for designing drugs to treatdiseases associated with altered mitochondrial function, including (byway of non-limiting theory) certain arthritic disorders, degenerativedisorders and hyperproliferative diseases. Further according tonon-limiting theory, depending on the disease or disorder for whichtreatment is sought, such drugs may be, for example, mitochondriaprotecting agents, anti-apoptotic agents or pro-apoptotic agents.

[0046] Clearly there is a need for compounds and methods that limit orprevent damage to organelles, cells and tissues that results directly orindirectly from mitochondrial dysfunction, for example damage by freeradicals generated intracellularly. In particular, because mitochondriaare essential organelles for producing metabolic energy, agents thatprotect mitochondria against such damage (e.g., oxidative injury by freeradicals) would be especially useful. Such agents may be suitable forthe treatment of degenerative diseases including mitochondria associateddiseases. Existing approaches to identifying agents that limit oxidativedamage may not include determination of whether such agents may helpprotect mitochondrial structure and/or function.

[0047] There is also a need for compounds and methods that limit orprevent damage to cells and tissues that occurs directly or indirectlyas a result of necrosis and/or inappropriate apoptosis. In particular,because mitochondria are mediators of apoptotic events, agents thatmodulate mitochondrially mediated pro-apoptotic events would beespecially useful. Such agents may be suitable for the treatment ofacute degenerative events such as stroke. Given the limited therapeuticwindow for blockade of necrotic death at the core of an infarct, it maybe particularly desirable to develop therapeutic strategies to limitneuronal death by preventing mitochondrial dysfunction in thenon-necrotic regions of an infarct. Agents and methods that maintainmitochondrial integrity during transient ischemia and the ensuing waveof excitotoxicity would be expected to be novel neuroprotective agentswith utility in limiting stroke-related neuronal injury.

[0048] There is also a need for compounds and methods that inhibit thegrowth or enhance the death of cells and tissues that have escapedappropriate apoptotic signals, as well as cytotoxic agents that causethe death of undesirable (e.g., cancer) cells by triggering theapoptotic cascade. In particular, because mitochondria are mediators ofapoptotic events, agents that stimulate mitochondrially mediatedpro-apoptotic events would be especially useful. Such agents may besuitable for the treatment of hyperproliferative diseases such as cancerand psoriasis.

[0049] The present invention fulfills these needs and provides otherrelated advantages. Those skilled in the art will recognize furtheradvantages and benefits of the invention after reading the disclosure.

SUMMARY OF THE INVENTION

[0050] Briefly stated, the present invention is directed to thetreatment of an arthritic disorder and/or to the treatment of a diseaseassociated with altered mitochondrial function by administration to awarm-blooded animal in need thereof an effective amount of a compoundhaving the following general structure (I):

[0051] including stereoisomers, prodrugs and pharmaceutically acceptablesalts thereof, wherein R₁ through R₅ are as defined below.

[0052] In certain embodiments, the invention provides a pharmaceuticalcomposition comprising an aryl N-cyanoguanidine compound of structure(I) and a pharmaceutically acceptable carrier. According to otherembodiments, the invention provides a method for treating an arthriticdisorder, by administering an effective amount of such a pharmaceuticalcomposition to an animal in need thereof. According to still furtherembodiments, there is provided a method for treating a diseaseassociated with altered mitochondrial function comprising administeringan effective amount of such a pharmaceutical composition to an animal inneed thereof.

[0053] These and other aspects of the present invention will becomeevident upon reference to the following detailed description. Inaddition, various references are set forth herein which describe in moredetail certain aspects of this invention, and are therefore incorporatedby reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWING

[0054]FIG. 1 illustrates the ability of a representative compound toblock SIN-1-mediated inhibition of mitochondrial respiration in TC28cells.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention provides compounds, compositions andmethods that are useful in treatment of arthritic disorders and/or ofdiseases associated with altered mitochondrial function. Morespecifically, the compounds of this invention have the followingstructure (I):

[0056] or a stereoisomer, prodrug or pharmaceutically acceptable saltthereof,

[0057] wherein

[0058] R₁, R₂, R₃, R₄ and R₅ are the same or different and individuallyhydrogen, halogen, hydroxy, alkyl, alkoxy, substituted alkyl, aryl,substituted aryl, arylalky, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl; or R₃ taken together with R₄, or R₄ taken togetherwith R₅, and further taken together with the respective carbon atom towhich these groups are attached, form an unsubstituted or substitutedfused aryl or heterocycle.

[0059] As used herein, the above terms have the following meanings:

[0060] “Alkyl” means a straight chain or branched, noncyclic or cyclic,unsaturated or saturated aliphatic hydrocarbon containing from 1 to 10carbon atoms, while the term “lower alkyl” has the same meaning as alkylbut contains from 1 to 6 carbon atoms. Representative saturated straightchain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl,n-hexyl, and the like; while saturated branched alkyls includeisopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, —CH₂cyclopropyl, —CH₂cyclobutyl,—CH₂cyclopentyl, —CH₂cyclohexyl, and the like; while unsaturated cyclicalkyls include cyclopentenyl and cyclohexenyl, and the like. Cyclicalkyls, also referred to as “homocyclic rings,” and include di- andpoly-homocyclic rings such as decalin and adamantyl. Unsaturated alkylscontain at least one double or triple bond between adjacent carbon atoms(referred to as an “alkenyl” or “alkynyl”, respectively). Representativestraight chain and branched alkenyls include ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike; while representative straight chain and branched alkynyls includeacetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl,3-methyl-1 butynyl, and the like.

[0061] “Aryl” means an aromatic carbocyclic moiety such as phenyl ornaphthyl.

[0062] “Arylalkyl” means an alkyl having at least one alkyl hydrogenatoms replaced with an aryl moiety, such as benzyl, —CH2-(1 or2-naphthyl), —(CH2)2phenyl, —(CH2)3phenyl, —CH(phenyl)2, and the like.

[0063] “Heteroaryl” means an aromatic heterocycle ring of 5- to 10members and having at least one heteroatom selected from nitrogen,oxygen and sulfur, and containing at least 1 carbon atom, including bothmono- and bicyclic ring systems. Representative heteroaryls include (butare not limited to) furyl, benzofuranyl, thiophenyl, benzothiophenyl,pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl,isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl,imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl,phthalazinyl, and quinazolinyl.

[0064] “Heteroarylalkyl” means an alkyl having at least one alkylhydrogen atom replaced with a heteroaryl moiety, such as —CH₂pyridinyl,—CH₂pyrimidinyl, and the like.

[0065] “Heterocycle” (also referred to herein as a “heterocycle ring”)means a 5- to 7-membered monocyclic, or 7- to 14-membered polycyclic,heterocycle ring which is either saturated, unsaturated or aromatic, andwhich contains from 1 to 4 heteroatoms independently selected fromnitrogen, oxygen and sulfur, and wherein the nitrogen and sulfurheteroatoms may be optionally oxidized, and the nitrogen heteroatom maybe optionally quaternized, including bicyclic rings in which any of theabove heterocycles are fused to a benzene ring as well as tricyclic (andhigher) heterocyclic rings. The heterocycle may be attached via anyheteroatom or carbon atom. Heterocycles include heteroaryls as definedabove. Thus, in addition to the aromatic heteroaryls listed above,heterocycles also include (but are not limited to) morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, and the like.

[0066] “Heterocyclealkyl” means an alkyl having at least one alkylhydrogen atom replaced with a heterocycle, such as —CH₂morpholinyl, andthe like.

[0067] The term “substituted” as used herein means any of the abovegroups (e.g., alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,heterocycle, heterocyclealkyl, etc.) wherein at least one hydrogen atomis replaced with a substituent. In the case of a keto substituent (“═O”)two hydrogen atoms are replaced. When substituted, “substituents” withinthe context of this invention include halogen, hydroxy, cyano, nitro,amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, —NR_(a)R_(b), —NR_(a)C(═O)R_(b),—NR_(a)C(═O)NR_(a)NR_(b), —NR_(a)C(═O)OR_(b) —NR_(a)SO₂R_(b), —OR_(a),—C(═O)R_(a) —C(═O)OR_(a), —C(═O)NR_(a)R_(b), —OC(═O)NR_(a)R_(b), —SH,—SR_(a), —SOR_(a), —S(═O)₂Ra, —OS(═O)₂Ra, —S(═O)₂OR_(a), wherein R_(a)and R_(b) are the same or different and independently hydrogen, alkyl,haloalkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substitutedheterocycle, heterocylealkyl or substituted heterocyclealkyl. Forexample, substituted alkyl includes trifluoromethyl.

[0068] “Halogen” means fluoro, chloro, bromo and iodo.

[0069] “Haloalkyl” means an alkyl having at least one hydrogen atomreplaced with halogen, such as trifluoromethyl and the like.

[0070] “Alkoxy” means an alkyl moiety attached through an oxygen bridge(i.e., —O-alkyl) such as methoxy, ethoxy, and the like.

[0071] In more specific embodiment of this invention, at least two of R₁through R₅ are hydrogen, an in another embodiment at least three of R₁through R₅ are hydrogen, and in still another embodiment at least fourof R₁ through R₅ are hydrogen.

[0072] In a further embodiment, R₁ through R₅ are the same or differentand independently hydrogen, alkyl, substituted alkyl, hydroxyl, halogenor alkoxy, wherein representative alkyl includes methyl, representativealkoxy includes methoxy and representative substituted alkyl includestrifluoromethyl.

[0073] In another embodiment, at least one of R₁ through R₅ is aheterocycle, such as morpholinyl.

[0074] In yet a further embodiment, R₃ taken together with R₄, or R₄taken together with R₅, and further taken together with the respectivecarbon atom to which these groups are attached, form an unsubstituted orsubstituted fused aryl or heterocycle. For example, in the case of anunsubstituted or substituted aryl, representative compounds of thisinvention have the following structure (II) when R₄ and R₅ takentogether form a fused aryl, and structure (III) when R₃ and R₄ takentogether form a fused aryl:

[0075] wherein the fused aryl portion of structure (II) or (III) may beoptionally substituted by one or more substituents as defined above.

[0076] The compounds of the present invention may be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. In general, the compounds of this invention maybe prepared by the following Reaction Scheme:

[0077] Reaction Scheme

[0078] In the above Reaction Scheme, N-cyano-S-methylisothiourea 1 isdissolved in i-PrOH, followed by addition of NaOH. The resultingsolution is heated and then cooled to generate the intermediate salt,sodium dicyanamide 2. This intermediate salt is then added to theappropriately-substituted analine 3 in HCl. The reaction mixture isheated, cooled and then evaporated to yield a compound of structure (I)as a crude product, which may then be purified to yield to a compound ofstructure (I) having the desired purity.

[0079] As noted above, clinical parameters and criteria for determiningthe presence or risk of an arthritic disorder are well established(e.g., Gilliland et al., “Disorders of the joints and connectivetissue,” Section 14, Harrison's Principles of Internal Medicine, EighthEd., Thorn et al., eds. McGraw-Hill, New York, N.Y., 1977, pp.2048-2080), as are criteria for determining the presence or risk of anumber of other diseases associated with altered mitochondrial function,as also provided herein (e.g., for AD, McKhann et al., Neurology 34:939,1984; DeKosky et al., Ann. Neurology 27(5):467-64, 1990; for diabetes,Gavin et al., Diabetes Care 22(suppl. 1):S5-S19, 1999; etc.—otherdiagnostic criteria for diseases associated with altered mitochondrialfunction will be familiar to those having ordinary skill in the art andbased on the disclosure herein). “Altered mitochondrial function” mayrefer to any condition or state, including those that may, according tonon-limiting theory, accompany an arthritic disorder, where anystructure or activity that is directly or indirectly related to amitochondrial function has been changed in a statistically significantmanner relative to a control or standard. Altered mitochondrial functionmay have its origin in extramitochondrial structures or events as wellas in mitochondrial structures or events, in direct interactions betweenmitochondrial and extramitochondrial genes and/or their gene products,or in structural or functional changes that occur as the result ofinteractions between intermediates that may be formed as the result ofsuch interactions, including metabolites, catabolites, substrates,precursors, cofactors and the like.

[0080] Additionally, altered mitochondrial function may include alteredrespiratory, metabolic or other biochemical or biophysical activity insome or all cells of a biological source. As non-limiting examples,markedly impaired ETC activity may be related to altered mitochondrialfunction, as may be generation of increased reactive oxygen species(ROS) or defective oxidative phosphorylation. As further examples,altered mitochondrial membrane potential (e.g., PCT/US99/22261;PCT/US00/17380), altered mitochondrial regulation of intracellularcalcium homeostasis (e.g., U.S. Pat. No. 6,140,067), induction ofapoptotic pathways and formation of a typical chemical and biochemicalcrosslinked species within a cell, whether by enzymatic or non-enzymaticmechanisms, may all be regarded as indicative of altered mitochondrialfunction. These and other non-limiting examples of altered mitochondrialfunction are described in greater detail below.

[0081] Without wishing to be bound by theory, altered mitochondrialfunction that may be characteristic of an arthritic disorder or ofanother disease associated with altered mitochondrial function, asprovided herein, may also be related to loss of mitochondrial membraneelectrochemical potential by mechanisms other than free radicaloxidation, for example by defects in transmitochondrial membraneshuttles and transporters such as the mitochondrial adenine nucleotidetransporter or the malate-aspartate shuttle, by intracellular calciumflux, by defects in ATP biosynthesis, by impaired association withmitochondrial porin (also known, e.g., as voltage dependent anionchannel, VDAC) of hexokinases or other enzymes or by other events. Suchcollapse of mitochondrial inner membrane potential may result fromdirect or indirect effects of mitochondrial genes, gene products orrelated downstream mediator molecules and/or extramitochondrial genes,gene products or related downstream mediators, or from other known orunknown causes.

[0082] By way of background, functional mitochondria contain geneproducts encoded by mitochondrial genes situated in mitochondrial DNA(mtDNA) and by extramitochondrial genes (e.g., nuclear genes) notsituated in the circular mitochondrial genome. The 16.5 kb mtDNA encodes22 tRNAs, two ribosomal RNAs (rRNA) and 13 enzymes of the electrontransport chain (ETC), the elaborate multi-complex mitochondrialassembly where, for example, respiratory oxidative phosphorylation takesplace. The overwhelming majority of mitochondrial structural andfunctional proteins are encoded by extramitochondrial, and in most casespresumably nuclear, genes. Accordingly, mitochondrial andextramitochondrial genes may interact directly, or indirectly via geneproducts and their downstream intermediates, including metabolites,catabolites, substrates, precursors, cofactors and the like. Alterationsin mitochondrial function, for example impaired electron transportactivity, defective oxidative phosphorylation or increased free radicalproduction, may therefore arise as the result of defective mtDNA,defective extramitochondrial DNA, defective mitochondrial orextramitochondrial gene products, defective downstream intermediates ora combination of these and other factors.

[0083] According to certain embodiments of the present invention, as itrelates to an arthritic disorder and/or a disease associated withaltered mitochondrial function, determination of altered (e.g.,increased or decreased in a statistically significant manner relative toa control) mitochondrial function may involve monitoring intracellularcalcium homeostasis and/or cellular responses to perturbations of thishomeostasis, including physiological and pathophysiological calciumregulation. In particular, according to these embodiments, a cellularresponse to elevated intracellular calcium in a biological sample from asubject known or suspected of having a disease associated with alteredmitochondrial function is compared to the response in a biologicalsample from a control subject. The range of cellular responses toelevated intracellular calcium is broad, as is the range of methods andreagents for the detection of such responses. Many specific cellularresponses are known to those having ordinary skill in the art; theseresponses will depend on the particular cell types present in a selectedbiological sample. As non-limiting examples, cellular responses toelevated intracellular calcium include secretion of specific secretoryproducts, exocytosis of particular pre-formed components, increasedglycogen metabolism and cell proliferation (see, e.g., Clapham, Cell80:259, 1995; Cooper, The Cell—A Molecular Approach, 1997 ASM Press,Washington, D.C.; Alberts, B., Bray, D., et al., Molecular Biology ofthe Cell, 1995 Garland Publishing, NY).

[0084] As a brief background, normal alterations of intramitochondrialCa²⁺ are associated with normal metabolic regulation (Dykens, 1998 inMitochondria & Free Radicals in Neurodegenerative Diseases, Beal, Howelland Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 29-55; Radi et al.,1998 in Mitochondria & Free Radicals in Neurodegenerative Diseases,Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 57-89;Gunter and Pfeiffer, Am. J. Physiol. 27:C755, 1991; Gunter et al., Am.J. Physiol. 267:313, 1994). For example, fluctuating levels ofmitochondrial free Ca²⁺ may be responsible for regulating oxidativemetabolism in response to increased ATP utilization, via allostericregulation of enzymes (reviewed by Crompton et al., Basic Res. Cardiol.88:513-23, 1993) and the glycerophosphate shuttle (Gunter et al., J.Bioenerg. Biomembr. 26:471, 1994).

[0085] Normal mitochondrial function includes regulation of cytosolicfree calcium levels by sequestration of excess Ca²⁺ within themitochondrial matrix. Depending on cell type, cytosolic Ca²⁺concentration is typically 50-100 nM. In normally functioning cells,when Ca²⁺ levels reach 200-300 nM, mitochondria begin to accumulate Ca²⁺as a function of the equilibrium between influx via a Ca²⁺ uniporter inthe inner mitochondrial membrane and Ca²⁺ efflux via both Na⁺ dependentand Na⁺independent calcium carriers. In certain instances, suchperturbation of intracellular calcium homeostasis is a feature ofdiseases associated with altered mitochondrial function, regardless ofwhether the calcium regulatory dysfunction is causative of, or aconsequence of, altered mitochondrial function.

[0086] Elevated mitochondrial calcium levels thus may accumulate inresponse to an initial elevation in cytosolic free calcium, as describedabove. Such elevated mitochondrial calcium concentrations in combinationwith reduced ATP or other conditions associated with mitochondrialpathology, can lead to collapse of mitochondrial inner membranepotential (see Gunter et al., Biochim. Biophys. Acta 1366:5, 1998;Rottenberg and Marbach, Biochim. Biophys. Acta 1016:87, 1990).Generally, the extramitochondrial (cytosolic) level of Ca²⁺ in abiological sample is greater than that present within mitochondria. Inthe case of a disease associated with altered mitochondrial function,mitochondrial or cytosolic calcium levels may vary from the above rangesand may range from, e.g., about 1 nM to about 500 mM, more typicallyfrom about 10 nM to about 100 μM and usually from about 20 nM to about 1μM, where “about” indicates ±10%. A variety of calcium indicators areknown in the art, including but not limited to, for example, fura-2(McCormack et al., Biochim. Biophys. Acta 973:420, 1989); mag-fura-2;BTC (U.S. Pat. No. 5,501,980); fluo-3, fluo-4 and fluo-5N (U.S. Pat. No.5,049,673); rhod-2; benzothiaza-1; and benzothiaza-2 (all of which areavailable from Molecular Probes, Eugene, Oreg.). These or any othermeans for monitoring intracellular calcium are contemplated fordetermining the presence of altered mitochondrial function (see, e.g.,PCT/US01/01500).

[0087] Thus, for determining altered mitochondrial function that ismanifest as a cellular response to elevated intracellular calcium,compounds that induce increased cytoplasmic and mitochondrialconcentrations of Ca²⁺, including calcium ionophores, are well known tothose of ordinary skill in the art, as are methods for measuringintracellular calcium (see, e.g., Gunter and Gunter, J. Bioenerg.Biomembr. 26:471, 1994; Gunter et al., Biochim. Biophys. Acta 1366:5,1998; McCormack et al., Biochim. Biophys. Acta 973:420, 1989; Orreniusand Nicotera, J. Neural. Transm. Suppl. 43:1, 1994; Leist and Nicotera,Rev. Physiol. Biochem. Pharmacol. 132:79, 1998; and Haugland, 1996,Handbook of Fluorescent Probes and Research Chemicals—Sixth Ed.,Molecular Probes, Eugene, Oreg.). Accordingly, a person skilled in theart may readily select a suitable ionophore (or another compound thatresults in increased cytoplasmic and/or mitochondrial concentrations ofCa²⁺) and an appropriate means for detecting intracellular calcium foruse in identifying altered mitochondrial function, according to theinstant disclosure and to well known methods.

[0088] Ca²⁺ influx into mitochondria appears to be largely dependent,and may be completely dependent, upon the negative transmembraneelectrochemical potential (ΔΨ) established at the inner mitochondrialmembrane by electron transfer, and such influx fails to occur in theabsence of AT even when an eight-fold Ca²⁺ concentration gradient isimposed (Kapus et al., 1991 FEBS Lett. 282:61). Accordingly,mitochondria may release Ca²⁺ when the membrane potential is dissipated,as occurs with uncouplers like 2,4-dinitrophenol and carbonyl cyanidep-trifluoro-methoxyphenylhydrazone (FCCP). Thus, according to certainembodiments of the present invention, collapse of AT may be potentiatedby influxes of cytosolic free calcium into the mitochondria, as mayoccur under certain physiological conditions including those encounteredby cells of a subject having an arthritic disorder. Detection of suchcollapse may be accomplished by a variety of means as provided herein.

[0089] In certain related embodiments of the invention, altered (e.g.,increased or decreased in a statistically significant manner relative toa control) mitochondrial membrane potential may be an indicator ofaltered mitochondrial function. Typically, mitochondrial membranepotential may be determined according to methods with which thoseskilled in the art will be readily familiar, including but not limitedto detection and/or measurement of detectable compounds such asfluorescent indicators, optical probes and/or sensitive pH andion-selective electrodes (see, e.g., Ernster et al., J. Cell Biol.91:227s, 1981; and references cited; see also Haugland, 1996 Handbook ofFluorescent Probes and Research Chemicals—Sixth Ed., Molecular Probes,Eugene, Oreg., pp. 266-274 and 589-594.). For example, by way ofillustration and not limitation, the fluorescent probes2-,4-dimethylaminostyryl-N-methylpyridinium (DASPMI) andtetramethylrhodamine esters (such as, e.g., tetramethylrhodamine methylester, TMRM; tetramethylrhodamine ethyl ester, TMRE) or relatedcompounds (see, e.g., Haugland, 1996, supra) may be quantified followingaccumulation in mitochondria, a process that is dependent on, andproportional to, mitochondrial membrane potential (see, e.g., Murphy etal., 1998 in Mitochondria & Free Radicals in Neurodegenerative Diseases,Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 159-186and references cited therein; and Molecular Probes On-line Handbook ofFluorescent Probes and Research Chemicals, athttp:/www.probes.com/handbook/toc.html). Other fluorescent detectablecompounds that may be used in the invention include but are not limitedto rhodamine 123, rhodamine B hexyl ester, DiOC₆(3), JC-1[5,5′,6,6′-Tetrachloro-1,1′,3,3′-TetraethylbezimidazolcarbocyanineIodide] (see Cossarizza, et al., Biochem. Biophys. Res. Comm. 197:40,1993; Reers et al., Meth. Enzymol. 260:406, 1995), rhod-2 (see U.S. Pat.No. 5,049,673; all of the preceding compounds are available fromMolecular Probes, Eugene, Oreg.) and rhodamine 800 (Lambda Physik, GmbH,Göttingen, Germany; see Sakanoue et al., J. Biochem. 121:29, 1997).Methods for monitoring mitochondrial membrane potential are alsodisclosed in U.S. application Ser. No. 09/161,172.

[0090] Mitochondrial membrane potential can also be measured bynon-fluorescent means, for example by using TTP (tetraphenylphosphoniumion) and a TTP-sensitive electrode (Kamo et al., J. Membrane Biol.49:105, 1979; Porter and Brand, Am. J. Physiol. 269:R1213, 1995). Thoseskilled in the art will be able to select appropriate detectablecompounds or other appropriate means for measuring ΔΨm. By way ofexample and not limitation, TMRM is somewhat preferable to TMRE because,following efflux from mitochondria, TMRE yields slightly more residualsignal in the endoplasmic reticulicum and cytoplasm than TMRM.

[0091] As another non-limiting example, membrane potential may beadditionally or alternatively calculated from indirect measurements ofmitochondrial permeability to detectable charged solutes, using matrixvolume and/or pyridine nucleotide redox determination combined withspectrophotometric or fluorimetric quantification. Measurement ofmembrane potential dependent substrate exchange-diffusion across theinner mitochondrial membrane may also provide an indirect measurement ofmembrane potential. (See, e.g., Quinn, 1976, The Molecular Biology ofCell Membranes, University Park Press, Baltimore, Md., pp. 200-217 andreferences cited therein.)

[0092] Exquisite sensitivity to extraordinary mitochondrialaccumulations of Ca²⁺ that result from elevation of intracellularcalcium, as described above, may also characterize a disease associatedwith altered mitochondrial function. Additionally, a variety ofphysiologically pertinent agents, including hydroperoxide and freeradicals, may synergize with Ca²⁺ to induce collapse of ΔΨ (Novgorodovet al., Biochem. Biophys. Acta 1058:242, 1991; Takeyama et al., Biochem.J. 294:719, 1993; Guidox et al., Arch. Biochem. Biophys. 306:139, 1993).Accordingly, non-limiting examples of methods for determining alteredmitochondrial function that is manifested in cellular responses toelevated intracellular calcium, or as altered mitochondrial membranepotential, include mitochondrial membrane potential (ΔΨ_(m)) assays(described in copending U.S. patent application Serial No. 60/140,433)and mitochondrial permeability transition (MPT) assays (described incopending U.S. patent application Ser. No. 09/161,172).

[0093] Altered mitochondrial function may also be determined bycomparing a cellular response to an apoptosis-inducing (“apoptogenic”)stimulus in a biological sample from (i) a subject believed to be atrisk for a disease associated with altered mitochondrial function, and(ii) a control subject. The range of cellular responses to various knownapoptogenic stimuli is broad, as is the range of methods and reagentsfor the detection of such responses. It is therefore within thecontemplation of the present invention to determine a disease associatedwith altered mitochondrial function by so comparing a cellular responseto an apoptogenic stimulus, where such response is an indicator ofaltered mitochondrial function as provided herein.

[0094] As noted above, mitochondrial dysfunction and/or related elevatedROS levels may initiate early events leading to apoptosis in variouscell types (Kroemer et al., FASEB J. 9:1277-87, 1995; Zamzami et al., J.Exp. Med. 182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72,1995; Ausserer et al., Mol. Cell. Biol. 14:5032-42, 1994). In severalcell types, reduction in the mitochondrial membrane potential (ΔΨm)precedes the nuclear DNA degradation that accompanies apoptosis. Incell-free systems, mitochondrial, but not nuclear, enriched fractionsare capable of inducing nuclear apoptosis (Newmeyer et al., Cell70:353-64, 1994). Perturbation of mitochondrial respiratory activityleading to altered cellular metabolic states, such as elevatedintracellular ROS, may occur in a disease associated with alteredmitochondrial function and may further induce pathogenetic events viaapoptotic mechanisms.

[0095] Oxidatively stressed mitochondria may release a pre-formedsoluble factor that can induce chromosomal condensation, an eventpreceding apoptosis (Marchetti et al., Cancer Res. 56:2033-38, 1996). Inaddition, members of the Bcl-2 family of anti-apoptosis gene productsare located within the outer mitochondrial membrane (Monaghan et al., J.Histochem. Cytochem. 40:1819-25, 1992) and these proteins appear toprotect membranes from oxidative stress (Korsmeyer et al, Biochim.Biophys. Act. 1271:63, 1995). Localization of Bcl-2 to this membraneappears to be indispensable for modulation of apoptosis (Nguyen et al.,J. Biol. Chem. 269:16521-24, 1994). Thus, changes in mitochondrialphysiology may be important mediators of apoptosis.

[0096] Altered mitochondrial function, as may be used to identify a riskfor a disease associated with altered mitochondrial function in asubject according to the present disclosure, may therefore lower thethreshold for induction of apoptosis by an apoptogen. A variety ofapoptogens are known to those familiar with the art (see, e.g., Green etal., Science 281:1309, 1998; and references cited therein) and mayinclude by way of illustration and not limitation apoptogens that, whenadded to cells under appropriate conditions with which those skilled inthe art will be familiar, require specific receptors such as the tumornecrosis factor, FasL, glutamate, NMDA, IL-1, IL-3, corticosterone,mineralcorticoid or glucocorticoid receptor(s). Apoptogens may furtherinclude herbimycin A (Mancini et al., J. Cell. Biol 138:449-69, 1997);paraquat (Costantini et al., Toxicology 99:1-2, 1995); ethylene glycols;protein kinase inhibitors such as, e.g.: staurosporine, calphostin C,caffeic acid phenethyl ester, chelerythrine chloride, genistein;1-(5-isoquinolinesulfonyl)-2-methylpiperazine;N-[2-((p-bromocinnamyl)amino)ethyl]-5-5-isoquinolinesulfonamide; KN-93;quercitin; d-erythro-sphingosine derivatives; UV radiation; ionophoressuch as, e.g., ionomycin, valinomycin and other ionophores known in theart; MAP kinase inducers such as, e.g., anisomycin and anandamine; cellcycle blockers such as, e.g., aphidicolin, colcemid, 5-fluorouracil andhomoharringtonine; acetylcholineesterase inhibitors such as, e.g.,berberine; anti-estrogens such as, e.g., tamoxifen; pro-oxidants, suchas, e.g., tert-butyl hydroperoxide, peroxynitrite, hydrogen peroxide andnitric oxide donors including but not limited to L-arginine,5,5′-dinitrosodithiol, N-hydroxy-L-arginine,S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, NOR-1, NOR-3,NOR4,4-phenyl-3-furoxancarbonitrile, 3-morpholinosydnonimine, sodiumnitroprusside and streptozotocin; glutathione depleting agents such as,e.g., ethacrynic acid (Meister, Biochim. Biophys. Acta. 1271:35, 1995);free radicals such as, e.g., nitric oxide; inorganic metal ions, suchas, e.g., cadmium; DNA synthesis inhibitors such as, e.g., actinomycinD; DNA intercalators such as, e.g., doxorubicin, bleomycin sulfate,hydroxyurea, methotrexate, mitomycin C, camptothecin, and daunorubicin;protein synthesis inhibitors such as, e.g., cycloheximide, puromycin,and rapamycin; agents that effect microtubule formation or stabilitysuch as, e.g.: vinblastine, vincristine, colchicine,4-hydroxyphenylretinamide, and paclitaxel; and other MPT inducers suchas, e.g., Bax protein (Jurgenmeier et al., PNAS 95:4997-5002, 1998),calcium and inorganic phosphate (Kroemer et al., Ann. Rev. Physiol.60:619, 1998).

[0097] Cells in a biological sample that are suspected of undergoingapoptosis may be examined for morphological, permeability or otherchanges that are indicative of an apoptotic state. For example by way ofillustration and not limitation, apoptosis in many cell types may causealtered morphological appearance such as plasma membrane blebbing, cellshape change, loss of substrate adhesion properties or othermorphological changes that can be readily detected by a person havingordinary skill in the art, for example by using light microscopy. Asanother example, cells undergoing apoptosis may exhibit fragmentationand disintegration of chromosomes, which may be apparent by microscopyand/or through the use of DNA-specific or chromatin-specific dyes thatare known in the art, including fluorescent dyes. Such cells may alsoexhibit altered plasma membrane permeability properties as may bereadily detected through the use of vital dyes (e.g., propidium iodide,trypan blue) or by the detection of lactate dehydrogenase leakage intothe extracellular milieu. These and other means for detecting apoptoticcells by morphologic criteria, altered plasma membrane permeability andrelated changes will be apparent to those familiar with the art.

[0098] Alternatively, where the indicator of altered mitochondrialfunction is a cellular response to an apoptogen, cells in a biologicalsample may be assayed for translocation of cell membranephosphatidylserine (PS) from the inner to the outer leaflet of theplasma membrane, which may be detected, for example, by measuring outerleaflet binding by the PS-specific protein annexin. (Martin et al., J.Exp. Med. 182:1545, 1995; Fadok et al., J. Immunol. 148:2207, 1992.) Instill another method for determining altered mitochondrial function bymonitoring a cellular response to an apoptogen, the cellular response tothe apoptogen is determined by an assay for induction of specificprotease activity in any member of a family of apoptosis-activatedproteases known as the caspases (see, e.g., Green et al., Science281:1309, 1998). Those having ordinary skill in the art will be readilyfamiliar with methods for determining caspase activity, for example bydetermination of caspase-mediated cleavage of specifically recognizedprotein substrates. These substrates may include, for example,poly-(ADP-ribose) polymerase (PARP) or other naturally occurring orsynthetic peptides and proteins cleaved by caspases that are known inthe art (see, e.g., Ellerby et al., J. Neurosci. 17:6165, 1997). Thesynthetic peptide Z-Tyr-Val-Ala-Asp-AFC (SEQ ID NO: ______;), wherein“Z” indicates a benzoyl carbonyl moiety and AFC indicates7-amino-4-trifluoromethylcoumarin (Kluck et al., Science 275:1132, 1997;Nicholson et al., Nature 376:37, 1995), is one such substrate. Othernon-limiting examples of substrates include nuclear proteins such asU1-70 kDa and DNA-PKcs (Rosen and Casciola-Rosen, J. Cell. Biochem.64:50, 1997; Cohen, Biochem. J. 326:1, 1997).

[0099] As described above, the mitochondrial inner membrane may exhibithighly selective and regulated permeability for many small solutes, butis impermeable to large (>˜10 kDa) molecules. (See, e.g., Quinn, 1976The Molecular Biology of Cell Membranes, University Park Press,Baltimore, Md.). In cells undergoing apoptosis, however, collapse ofmitochondrial membrane potential may be accompanied by increasedpermeability permitting macromolecule diffusion across the mitochondrialmembrane. Thus, in another method for assaying a cellular response to anapoptogen, detection of a mitochondrial protein, for example cytochromec or an intermembrane space protein, that has escaped from mitochondriain apoptotic cells may provide evidence of a response to an apoptogenthat can be readily determined. (Liu et al., Cell 86:147, 1996.) Suchdetection of cytochrome c may be performed spectrophotometrically,immunochemically or by other well established methods for determiningthe presence of a specific protein.

[0100] For instance, release of cytochrome c from cells challenged withapoptotic stimuli (e.g., ionomycin, a well known calcium ionophore) canbe followed by a variety of immunological methods. Matrix-assisted laserdesorption ionization time-of-flight (MALDI-TOF) mass spectrometrycoupled with affinity capture is particularly suitable for such analysissince apo-cytochrome c and holo-cytochrome c can be distinguished on thebasis of their unique molecular weights. For example, theSurface-Enhanced Laser Desorption/Ionization (SELDI™) system (Ciphergen,Palo Alto, Calif.) may be utilized to detect cytochrome c release frommitochondria in apoptogen treated cells. In this approach, a cytochromec specific antibody immobilized on a solid support is used to capturereleased cytochrome c present in a soluble cell extract. The capturedprotein is then encased in a matrix of an energy absorption molecule(EAM) and is desorbed from the solid support surface using pulsed laserexcitation. The molecular mass of the protein is determined by its timeof flight to the detector of the SELDI™ mass spectrometer.

[0101] A person having ordinary skill in the art will readily appreciatethat there may be other suitable techniques for quantifying apoptosis,and such techniques for purposes of determining altered mitochondrialfunction as manifested in a cellular response to an apoptogenic stimulusare within the scope of the methods provided by the present invention.

[0102] Detection of free radical production in a biological sample mayalso be employed to determine the presence of altered mitochondrialfunction, in a biological sample from a subject. Although mitochondriaare a primary source of free radicals in biological systems (see, e.g.,Murphy et al., 1998 in Mitochondria and Free Radicals inNeurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds.,Wiley-Liss, New York, pp. 159-186 and references cited therein), theinvention should not be so limited and free radical production can be anindicator of altered mitochondrial function regardless of the particularsubcellular source site. For example, numerous intracellular biochemicalpathways that lead to the formation of radicals through production ofmetabolites such as hydrogen peroxide, nitric oxide or superoxideradical via reactions catalyzed by enzymes such as flavin-linkedoxidases, superoxide dismutase or nitric oxide synthetase, are known inthe art, as are methods for detecting such radicals (see, e.g., Kelver,Crit. Rev. Toxicol. 23:21, 1993; Halliwell B. et al., Free Radicals inBiology and Medicine, 1989, Clarendon Press, Oxford, UK; Davies, K. J.A. et al., The Oxygen Paradox, Cleup Univ. Press, Padova, IT). Alteredmitochondrial function, such as failure at any step of the ETC, may alsolead to the generation of highly reactive free radicals. As noted above,radicals resulting from altered mitochondrial function include reactiveoxygen species (ROS), for example, superoxide, peroxynitrite andhydroxyl radicals, and potentially other reactive species that may betoxic to cells. Accordingly, in certain preferred embodiments of theinvention an indicator of altered mitochondrial function may be adetectable free radical species present in a biological sample. Incertain particularly preferred embodiments, the detectable free radicalwill be a ROS.

[0103] Methods for detecting a free radical that may be useful as anindicator of altered mitochondrial function are known in the art andwill depend on the particular radical. Typically, a level of freeradical production in a biological sample may be determined according tomethods with which those skilled in the art will be readily familiar,including but not limited to detection and/or measurement of:glycoxidation products including pentosidine, carboxymethylysine andpyrroline; lipoxidation products including glyoxal, malondialdehyde and4-hydroxynonenal; thiobarbituric acid reactive substances (TBARS; see,e.g., Steinbrecher et al., Proc. Nat. Acad. Sci. USA 81:3883, 1984;Wolff, Br. Med. Bull. 49:642, 1993) and/or other chemical detectionmeans such as salicylate trapping of hydroxyl radicals (e.g., Ghiselliet al., Meths. Mol. Biol. 108:89, 1998; Halliwell et al., Free Radic.Res. 27:239, 1997) or specific adduct formation (see, e.g., Mecocci etal., Ann. Neurol. 34:609, 1993; Giulivi et al., Meths. Enzymol. 233:363,1994) including malondialdehyde formation, protein nitrosylation, DNAoxidation including mitochondrial DNA oxidation, 8′-OH-guanosine adducts(e.g., Beckman et al., Mutat. Res. 424:51, 1999), protein oxidation,protein carbonyl modification (e.g., Baynes et al., Diabetes 40:405,1991; Baynes et al., Diabetes 48:1, 1999); electron spin resonance (ESR)probes; cyclic voltametry; fluorescent and/or chemiluminescentindicators (see also e.g., Greenwald, R. A. (ed.), Handbook of Methodsfor Oxygen Radical Research, 1985, CRC Press, Boca Raton, Fla.; Acworthand Bailey, (eds.), Handbook of Oxidative Metabolism, 1995, ESA, Inc.,Chelmsford, Mass.; Yla-Herttuala et al., J. Clin. Invest. 84:1086, 1989;Velazques et al., Diabetic Medicine 8:752, 1991; Belch et al., Int.Angiol. 14:385, 1995; Sato et al., Biochem. Med. 21:104, 1979; Traversoet al., Diabetologia 41:265, 1998; Haugland, 1996 Handbook ofFluorescent Probes and Research Chemicals—Sixth Ed., Molecular Probes,Eugene, Oreg., pp. 483-502, and references cited therein). For example,by way of illustration and not limitation, oxidation of the fluorescentprobes dichlorodihydrofluorescein diacetate and its carboxylatedderivative carboxydichlorodihydrofluorescein diacetate (see, e.g,Haugland, 1996, supra) may be quantified following accumulation incells, a process that is dependent on, and proportional to, the presenceof reactive oxygen species (see also, e.g., Molecular Probes On-lineHandbook of Fluorescent Probes and Research Chemicals, athttp://www.probes.com/handbook/toc.html). Other fluorescent detectablecompounds that may be used in the invention for detection of freeradical production include but are not limited to dihydrorhodamine anddihydrorosamine derivatives, cis-parinaric acid, resorufin derivatives,lucigenin and any other suitable compound that may be known to thosefamiliar with the art.

[0104] Thus, as also described above, free radical mediated damage mayinactivate one or more of the myriad proteins of the ETC and in doingso, may uncouple the mitochondrial chemiosmotic mechanism responsiblefor oxidative phosphorylation and ATP production. Indicators of alteredmitochondrial function that are ATP biosynthesis factors, includingdetermination of ATP production, are described in greater detail, forexample, in PCT/US00/25317 and in U.S. Pat. No. 6,140,067. Free radicalmediated damage to mitochondrial functional integrity is also just oneexample of multiple mechanisms associated with altered mitochondrialfunction that may result in collapse of the electrochemical potentialmaintained by the inner mitochondrial membrane. Methods for detectingchanges in the inner mitochondrial membrane potential are describedabove and in co-pending U.S. patent application Ser. No. 09/161,172.

[0105] Biological samples may comprise any tissue or cell preparation inwhich at least one candidate indicator of altered mitochondrial functioncan be detected, and may vary in nature accordingly, depending on theparticular indicator(s) to be compared. Thus, as will be apparent tothose having ordinary skill in the art based on the disclosure herein,in certain highly preferred embodiments biological samples comprisecells or cell preparations containing mitochondria, and in certain otherpreferred embodiments biological samples may comprise submitochondrialparticles. Biological samples may be provided by obtaining a bloodsample, biopsy specimen, tissue explant, organ culture or any othertissue or cell preparation from a subject or a biological source. Thesubject or biological source may be a human or non-human animal, aprimary cell culture or culture adapted cell line including but notlimited to genetically engineered cell lines that may containchromosomally integrated or episomal recombinant nucleic acid sequences,immortalized or immortalizable cell lines, somatic cell hybrid orcytoplasmic hybrid “cybrid” cell lines, differentiated ordifferentiatable cell lines, transformed cell lines and the like. Inparticularly preferred embodiments the subject or biological source is ahuman or non-human vertebrate, and in other particularly preferredembodiments the subject or biological source is a vertebrate-derivedprimary cell culture or culture-adapted cell line as provided herein,but the invention need not be so limited. As a non-limiting example byway of illustration, in certain embodiments the invention contemplates abiological sample that may be a non-vertebrate tissue or cellpreparation that has been artificially manipulated, for example throughrecombinant genetic engineering, to contain one or morevertebrate-derived genes, gene products or the like, such asmitochondrial molecular components and/or ATP biosynthesis factors asprovided, for example, in PCT/US00/25317 and in U.S. Pat. No. 6,140,067.For instance, a number of yeast and insect cell lines may be readilyreconstituted with heterologous vertebrate-derived components accordingto established methods with which those skilled in the art will befamiliar, to generate a model system for a disease associated withaltered mitochondrial function as provided herein. Accordingly, numerousvariations and modifications to biological samples are within thecontemplated scope and spirit of the present invention.

[0106] In certain other particularly preferred embodiments of theinvention, the subject or biological source may be suspected of havingor being at risk for having an arthritic disorder and/or a diseaseassociated with altered mitochondrial function, and in certain preferredembodiments of the invention the subject or biological source may beknown to be free of a risk or presence of such a disease. In certainother preferred embodiments where it is desirable to determine whetheror not a subject or biological source falls within clinical parametersindicative of an arthritic disorder, signs and symptoms of an arthriticdisorder that are accepted by those skilled in the art may be used to sodesignate a subject or biological source, for example clinical signsreferred to in Primer on the Rheumatic Diseases (7^(th) Edition, J. H.Klippel (ed.), 1997 The Arthritis Foundation, Atlanta, Ga.) andreferences cited therein, or other means known in the art for diagnosingan arthritic disorder. Similarly, clinical parameters indicative ofcertain other diseases associated with altered mitochondrial function asprovided herein are known to the art and are discussed above.

[0107] In certain embodiments of the invention, biological samples froma subject or biological source in which at least one alteredmitochondrial function has been detected may be compared before andafter contacting the subject or biological source with a composition ofstructure (I) such as an aryl N-cyanoguanidine agent as provided herein,for example to identify a candidate mitochondrial function in which theagent is capable of effecting a change, relative to the level of themitochondrial function before exposure of the subject or biologicalsource to the agent.

[0108] In a most preferred embodiment of the invention, the biologicalsample containing in which altered mitochondrial function is determinedcomprises a chondrocyte, and still more preferably, an articularchondrocyte. Chondrocytes can be obtained, for example, from normalmature cartilage tissue. For instance, U.S. Pat. Nos. 4,846,835 and5,041,138 disclose isolation of chondrocytes by digesting articularcartilage in a collagenase solution, followed by mitotic expansion ofthe chondrocytes in vitro. In another preferred embodiment of theinvention, the biological sample containing at least one candidateindicator of altered mitochondrial function may comprise a matrixvesicle (MV) derived from a chondrocyte (e.g., Anderson, Rheum. Dis.Clin. North Amer. 14:303, 1988; Doyle, J. Pathol. 136:199, 1982;Doherty, Hosp. Pract. Off. Ed. 29:93, 1994), for example, an MV preparedaccording to any of a number of established procedures (e.g., Johnson etal., J. Bone Miner. Res. 14:883, 1999) or by other techniques with whichthose having ordinary skill in the art will be familiar.

[0109] The initiation of matrix calcification by chondrocytes, as wellas by osteoblasts, appears to be mediated by the release ofmembrane-limited cell fragments known as matrix vesicles (MVs). MVcomponents, including a variety of enzymes, modify the extracellularmatrix, and the MV interiors serve as a sheltered environment forhydroxyapatite crystal formation (Anderson, Clin. Orthopaed. Rel. Res.314:266-80, 1995; Boskey et al., Calcif. Tissue Int. 60:309-15, 1997;Boskey, Connect. Tissue Res. 35:357-63, 1996; and Goldberg, Prog.Histochem. Cytochem. 31:1-187, 1996). Methods of preparing MVs aredescribed herein, and other methods are known in the art (see, e.g.,Johnson et al., J. Bone Miner. Res. 14:883-92, 1999, and U.S. Pat. No.5,656,450).

[0110] Mitochondria and SMPs can be prepared by a variety of methods(see, e.g., Fleischer et al., Methods Enzymol. 31:292-99, 1974; Pedersenet al., Methods Cell. Biol. 20:411-81, 1978; della-Cioppa et al., Mol.Cell. Endocrinol. 48:111-20, 1986; and Lauquin et al., Biochim. Biophys.Acta 460:331-45, 1977). For example, to prepare mitochondria and/orSMPs, the following procedure may be used. Cell lysates are centrifugedat 600×g for 10 minutes at 4° C., and this first supernatant is removedand set aside. The pellet, which comprises plasma membrane material, iswashed with 100 μl of MSB (210 mM mannitol, 70 mM sucrose, 50 mMTris-HCl, pH 7.4, and 10 mM EDTA) and centrifuged at 600×g for 10minutes at 4° C., in order to produce a second supernatant. The firstand second supernatants are combined and centrifuged at 14,000×g for 15minutes at 4° C.; the resultant pellet represents a mitochondrialfraction that is resuspended in MSB in order to prepare mitochondria.Such mitochondria may be incubated with 0.25 mg/ml digitonin (RocheMolecular Biochemicals, Indianapolis, Ind.) for 2 minutes and sonicatedfor 3 minutes at 50% duty cycle in a cup-horn sonicator to producesubmitochondrial particles (SMPs).

[0111] Accordingly, a biological sample as provided herein may incertain preferred embodiments comprise a chondrocyte,chondrocyte-derived MVs and/or chondrocyte-derived submitochondrialparticles (SMP), in which levels of one or more indicators of alteredmitochondrial function may be compared.

[0112] In another preferred embodiment of the invention, the biologicalsample containing at least one candidate indicator of alteredmitochondrial function may comprise whole blood, and may in anotherpreferred embodiment comprise a crude buffy coat fraction of wholeblood, which is known in the art to comprise further a particulatefraction of whole blood enriched in platelets and in nucleated bloodcells (e.g., white blood cells such as lymphocytes, monocytes andgranulocytes including neutrophils, eosinophils and basophils), andsubstantially depleted of erythrocytes. Those familiar with the art willknow how to prepare such a buffy coat fraction, which may be prepared,for example, by differential density sedimentation of blood componentsunder defined conditions, including the use of density dependentseparation media, or by other methods. In other preferred embodiments,the biological sample containing at least one indicator of alteredmitochondrial function may comprise an enriched, isolated or purifiedblood cell subpopulation fraction such as, for example, lymphocytes,polymorphonuclear leukocytes, granulocytes and the like. Methods for theselective preparation of particular hematopoietic cell subpopulationsare well known in the art (see, e.g., Current Protocols in Immunology,J. E. Coligan et al., (Eds.) 1998, John Wiley & Sons, NY).

[0113] According to certain embodiments of the invention, the particularcell type or tissue type from which a biological sample is obtained mayinfluence qualitative or quantitative aspects of at least one candidateindicator of altered mitochondrial function contained therein, relativeto the corresponding candidate indicator of altered mitochondrialfunction obtained from distinct cell or tissue types of a commonbiological source. It is therefore within the contemplation of theinvention to quantify at least one candidate indicator of alteredmitochondrial function in biological samples from different cell ortissue types as may render the advantages of the invention most usefulfor a particular indication, for example, an arthritic disorder or adisease associated with altered mitochondrial function as providedherein, and further for a particular degree of progression of a known orsuspected arthritic disorder (or disease associated with alteredmitochondrial function) in a vertebrate subject. The relevant cell ortissue types will be known to those familiar with such diseases.

[0114] For example, as provided herein, articular cartilage chondrocytesmay represent a particularly preferred cell type in the context of anarthritic disorder, as also may other cell types in joint development,stabilization, maintenance and repair processes such as cartilagehomeostasis, bone or ligament graft healing, scar tissue resorption orconnective tissue remodeling, for example, bone cells, osteoblasts,osteoclasts, bone marrow stromal cells, myocytes, motor nerve/end platecells, inflammatory cells and/or synoviocytes.

[0115] In order to determine whether a mitochondrial alteration maycontribute to a particular disease state, it may be useful to constructa model system for diagnostic tests and for screening candidatetherapeutic agents in which the nuclear genetic background may be heldconstant while the mitochondrial genome is modified. It is known in theart to deplete mitochondrial DNA from cultured cells to produce ρ⁰cells, thereby preventing expression and replication of mitochondrialgenes and inactivating mitochondrial function. It is further known inthe art to repopulate such ρ⁰ cells with mitochondria derived fromforeign cells in order to assess the contribution of the donormitochondrial genotype to the respiratory phenotype of the recipientcells. Such cytoplasmic hybrid cells, containing genomic andmitochondrial DNAs of differing biological origins, are known ascybrids. See, for example, International Publication Number WO 95/26973and U.S. Pat. No. 5,888,498 which are hereby incorporated by referencein their entireties, and references cited therein.

[0116] In certain other embodiments, the invention provides a method oftreating a patient having an arthritic disorder by administering to thepatient a composition comprising an agent having chemical structure (I)that substantially improves (e.g., alters to be closer to a control orasymptomatic state in a statistically significant manner) at least oneclinical criterion for having or being at risk for having an arthriticdisorder (see, e.g., Primer on the Rheumatic Diseases, 7^(th) Edition,J. H. Klippel (ed.), 1997 The Arthritis Foundation, Atlanta, Ga.). Theinvention also provides a method of treating a patient having a diseaseassociated with altered mitochondrial function by administering to thepatient a composition comprising an agent having chemical structure (I)that substantially improves (e.g., alters to be closer to a control orasymptomatic state in a statistically significant manner) at least oneclinical criterion for having or being at risk for having such adisease, as known in the art and as provided herein. Those havingordinary skill in the art can readily determine whether a change in suchclinical criterion brings that level closer to a normal value and/orclinically benefits the subject. Thus, a preferred agent provided by thepresent invention may include an agent capable of fully or partiallyrestoring such level.

[0117] Accordingly, in certain preferred embodiments as provided herein,a pharmaceutical composition suitable for treating an arthritic disorderand/or for treating a disease associated with altered mitochondrialfunction comprises an agent of structure (I), e.g., an arylN-cyanoguanidine agent. In the case of arthritic disorders, such agentsmay be used to prevent or treat arthritic disorders, such asosteoarthritis, degenerative joint disease and the like, and to promotethe healing of injured cartilage, for example, cartilage damaged bytrauma or repetitive motion disorder. Without wishing to be bound by anyparticular theory, some such agents may have activity as antioxidantsand presumably act by preventing or ameriolating the effects ofoxidative stress damage to mitochondria (for a review, see, e.g.,Kowaltowski et al., Free Radical Biol. Med. 26:463-471, 1999). Theseand/or other such agents may act to prevent programmed cell death(apoptosis), which may contribute to the development of osteoarthritis(Blanco et al., Arthritis & Rheumatism 41:284-289, 1998) and/or to otherdiseases associated with altered mitochondrial function as providedherein, or may exert clinically beneficial effects through othermechanisms.

[0118] Thus, within these and other related embodiments, a compositioncomprising structure (I) (e.g., an aryl N-cyanoguanidine agent) such asthose provided herein may be administered to a patient for treatment orprevention of an arthritic disorder or a disease associated with alteredmitochondrial function as provided herein. In certain preferredembodiments the agent is therefore a mitochondrial function-alteringagent. Therapeutic agents provided herein are preferably part of apharmaceutical composition when used in the methods of the presentinvention. The pharmaceutical composition will include at least one of apharmaceutically acceptable carrier, diluent or excipient, in additionto one or more mitochondrial function-altering agents and, optionally,other components.

[0119] A compound according to this invention (e.g., a composition ofstructure (I) such as an aryl N-cyanoguanidine agent), or apharmaceutically acceptable salt thereof, is administered to a patientin a therapeutically effective amount. A therapeutically effectiveamount is an amount calculated to achieve the desired effect. It will beapparent to one skilled in the art that the route of administration mayvary with the particular treatment. Routes of administration may beeither non-invasive or invasive. Non-invasive routes of administrationinclude oral, buccal/sublingual, rectal, nasal, topical (includingtransdermal and ophthalmic), vaginal, intravesical, and pulmonary.Invasive routes of administration include intraarterial, intravenous,intradermal, intramuscular, subcutaneous, intraperitoneal, intrathecaland intraocular.

[0120] The required dosage may vary with the particular treatment androute of administration. In general, dosages for compounds of thisinvention such as aryl N-cyanoguanidine agents of structure (I) asdescribed herein will be from about 1 to about 5 milligrams of thecompound per kilogram of the body weight of the host animal per day;frequently it will be between about 100 μg and about 5 mg but may varyup to about 50 mg of compound per kg of body weight per day. Therapeuticadministration is generally performed under the guidance of a physician,and pharmaceutical compositions contain the agent in a pharmaceuticallyacceptable carrier. These carriers are well known in the art andtypically contain non-toxic salts and buffers. Such carriers maycomprise buffers like physiologically-buffered saline,phosphate-buffered saline, carbohydrates such as glucose, mannose,sucrose, mannitol or dextrans, amino acids such as glycine,antioxidants, chelating agents such as EDTA or glutathione, adjuvantsand preservatives. Acceptable nontoxic salts include acid addition saltsor metal complexes, e.g., with zinc, iron, calcium, barium, magnesium,aluminum or the like (which are considered as addition salts forpurposes of this application). Illustrative of such acid addition saltsare hydrochloride, hydrobromide, sulphate, phosphate, tannate, oxalate,fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate,succinate, malate, ascorbate, tartrate and the like. If the activeingredient is to be administered in tablet form, the tablet may containa binder, such as tragacanth, corn starch or gelatin; a disintegratingagent, such as alginic acid; and a lubricant, such as magnesiumstearate. If administration in liquid form is desired, sweetening and/orflavoring may be used, and intravenous administration in isotonicsaline, phosphate buffer solutions or the like may be effected.

[0121] In one embodiment of the invention, pharmaceutical compositionscomprising one or more compounds of this invention are entrapped withinliposomes. Liposomes are microscopic spheres having an aqueous coresurrounded by one or more outer layer(s) made up of lipids arranged in abilayer configuration (see, e.g., Chonn et al., Current Op. Biotech.6:698, 1995). The therapeutic potential of liposomes as drug deliveryagents was recognized nearly thirty years ago (Sessa et al., J. LipidRes. 9:310, 1968). Liposomes include “sterically stabilized liposome,” aterm which, as used herein, refers to a liposome comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters 223:42, 1987; Wu et al., Cancer Research 53:3765,1993).

[0122] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 507:64, 1987)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A. 85:6949, 1988). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0123] Various liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn. 53:2778, 1980) describedliposomes comprising a nonionic detergent, 2C₁₂15G, that contains a PEGmoiety. Illum et al. (FEBS Letters 167:79, 1984) noted that hydrophiliccoating of polystyrene particles with polymeric glycols results insignificantly enhanced blood half-lives. Synthetic phospholipidsmodified by the attachment of carboxylic groups of polyalkylene glycols(e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and4,534,899). Klibanov et al. (FEBS Letts. 268:235, 1990) describedexperiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta 1029:91, 1990) extended such observationsto other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from thecombination of distearoylphosphatidylethanolamine (DSPE) and PEG.Liposomes having covalently bound PEG moieties on their external surfaceare described in European Patent No. 0 445 131 B1 and WO 90/04384 toFisher. Liposome compositions containing 1-20 mole percent of PEderivatized with PEG, and methods of use thereof, are described byWoodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al.(U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1).Liposomes comprising a number of other lipid-polymer conjugates aredisclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin etal.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

[0124] Compounds of the present invention (e.g., compositions ofstructure (I) such as aryl N-cyanoguanidine agents) as provided by thepresent invention also include prodrugs thereof. As used herein, a“prodrug” is any covalently bonded carrier that releases in vivo theactive parent drug when such prodrug is administered to a vertebratesubject. Prodrugs of a given compound are prepared by modifyingfunctional groups present on the compound in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound. Prodrugs include, but are not limited to, compoundswherein hydroxy, amine or sulfhydryl groups are bonded to any group ofthe parent compound via a bond that, when the prodrug is administered toa subject, cleaves to form the free hydroxyl, amino or sulfhydryl group,respectively. Representative examples of prodrugs include, but are notlimited to, acetate, formate and benzoate derivatives of alcohol andamine functional groups.

[0125] “Pharmaceutically acceptable carriers” for therapeutic use arewell known in the pharmaceutical art, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andeven flavoring agents may be provided in the pharmaceutical composition.For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. In addition, antioxidants andsuspending agents may be used. Optionally, for certain routes ofadministration, an anesthetic may be included in the formulation.

[0126] Pharmaceutically acceptable salts of the compounds of thisinvention may be made by techniques well known in the art, such as byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water of in anorganic solvent. Suitable salts in this context may be found inRemington's Pharmaceutical Sciences, 17^(th) ed., Mack Publishing Co.,Easton, Pa., 1985, which is hereby incorporated by reference.

[0127] By way of example and not limitation, suitable pharmaceuticallyacceptable salts of the compounds of this invention include acidaddition salts which may, for example, be formed by mixing a solution ofthe compound according to the invention with a solution of an acceptableacid such as hydrobromic acid, hydrochloric acid, fumaric acid, oxalicacid, p-toluenesulphonic acid, malic acid, maleic acid, methanesulfonicacid, succinic acid, acetic acid, citric acid, tartaric acid, carbonicacid, phosphoric acid, sulphuric acid and the like. The salts may beformed by conventional means, such as by reacting the free base form ofthe product with one or more equivalents of the appropriate acid in asolvent or medium in which the salt is insoluble, or in a solvent suchas water which is removed in vacuo or by freeze drying or by exchangingthe anions of an existing salt for another anion on a suitable ionexchange resin. By way of example and not limitation, suitablepharmaceutically acceptable salts of the compounds of this inventioninclude acid addition salts which may, for example, be formed by mixinga solution of the compound according to the invention with a solution ofan acceptable acid such as hydrobromic acid, hydrochloric acid, fumaricacid, oxalic acid, p-toluenesulphonic acid, malic acid, maleic acid,methanesulfonic acid, succinic acid, acetic acid, citric acid, tartaricacid, carbonic acid, phosphoric acid, sulphuric acid and the like. Thesalts may be formed by conventional means, such as by reacting the freebase form of the product with one or more equivalents of the appropriateacid in a solvent or medium in which the salt is insoluble, or in asolvent such as water which is removed in vacuo or by freeze drying orby exchanging the anions of an existing salt for another anion on asuitable ion exchange resin.

[0128] The pharmaceutical compositions that contain one or morecompounds of the invention as disclosed herein may be in any form whichallows for the composition to be administered to a patient. For example,the composition may be in the form of a solid, liquid or gas (aerosol).Typical routes of administration include, without limitation, oral,topical, parenteral (e.g., sublingually or buccally), sublingual,rectal, vaginal, and intranasal. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intrasternal, intrathecal, intracavernous, intrameatal, intraurethralinjection or infusion techniques. The pharmaceutical composition isformulated so as to allow the active ingredients contained therein to bebioavailable upon administration of the composition to a patient.Compositions that will be administered to a patient take the form of oneor more dosage units, where for example, a tablet may be a single dosageunit, and a container of one or more compounds of the invention inaerosol form may hold a plurality of dosage units.

[0129] For oral administration, an excipient and/or binder may bepresent. Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed. Thecomposition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain, inaddition to one or more compounds of structure (I), one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

[0130] A liquid pharmaceutical composition as used herein, whether inthe form of a solution, suspension or other like form, may include oneor more of the following adjuvants: sterile diluents such as water forinjection, saline solution, preferably physiological saline, Ringer'ssolution, isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

[0131] A liquid composition intended for either parenteral or oraladministration should contain an amount of a compound of structure (I)such that a suitable dosage will be obtained. Typically, this amount isat least 0.01 wt % of a compound of the invention in the composition.When intended for oral administration, this amount may be varied to bebetween 0.1 and about 70% of the weight of the composition. Preferredoral compositions contain between about 4% and about 50% of the compoundof the invention. Preferred compositions and preparations are preparedso that a parenteral dosage unit contains between 0.01 to 1% by weightof active compound.

[0132] The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the compound of the invention of from about 0.1 toabout 10% w/v (weight per unit volume).

[0133] The composition may be intended for rectal administration, in theform, e.g., of a suppository that will melt in the rectum and releasethe drug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

[0134] In certain preferred methods of the invention, the compound(s) ofthe invention may be administered through use of insert(s), bead(s),timed-release formulation(s), patch(es) or fast-release formulation(s).It will be evident to those of ordinary skill in the art that theoptimal dosage of the agent(s) may depend on the weight and physicalcondition of the patient; on the severity and longevity of the physicalcondition being treated; on the particular form of the activeingredient, the manner of administration and the composition employed.It is to be understood that use of the compounds of the presentinvention in chemotherapy can involve such an agent being bound toanother compound, for example, a monoclonal or polyclonal antibody, aprotein or a liposome, which assist the delivery of said compound.

[0135] These and related advantages will be appreciated by thosefamiliar with the art. The following Examples are offered by way ofillustration and not limitation.

EXAMPLES Example 1 GENERAL SYNTHESIS OF REPRESENTATIVE COMPOUNDS

[0136] N-Cyano-S-methylisothiourea (1.38 g, 12.0 mmol) was dissolved ini-PrOH (18.0 mL). To this stirred solution was added aqueous NaOH (2.0M, 6.0 mL), and the resulting reaction mixture was heated at 100° C. for30 min. The solution was allowed to cool down to ambient temperature,and 2.0 ml portions (each containing ca 1.0 mmol of the putativeintermediate salt, sodium dicyanamide) were added to a solution of theappropriate aniline (1.0 mmol) in HCl_((aq)) (1.0 M, 1.0 mL). Thereaction mixture was heated at 100° C. for 60 min with agitation.

[0137] After cooling down to room temperature, the reaction mixture wasevaporated under reduced pressure furnishing the crude product ofstructure (I). Prior to purification, each crude mixture was taken up inMeOH (10 mL), sonicated to break up solids, and filtered through 0.20micron PTFE membrane filters. Preparative RP-HPLC was performed on anautomated Gilson 215 HPLC system, each derivative being purified inthree batches (3.3 mL injection volumes) over a Betasil™ C18 column(150×20 mm, 5 μparticles, 100 Å pores, Keystone Scientific, Inc.,Bellefonte, Pa.). The product was eluted using a gradient of MeCN:TFA(10000:5) in H₂O:TFA (10000:5) at a flowrate of 15.0 mL/min. Appropriatefractions were analyzed for presence of desired product by LC/MS. Thepooled fractions were concentrated and repeatedly co-evaporated withMeOH (3×5.0 mL). LC/MS and NMR analyses were used for final confirmationof structure. Yields were in the range 10-65%.

[0138] The representative compounds made by this procedure, along withcorresponding analytical data, are summarized in the following Table 1.TABLE 1 Representative Compounds (I)

Cpd.

ESI-MS [M + H]⁺(calculated/observed) ¹H-NMR (1)

191.1/191.2 (d₆-DMSO) 9.35 (s, 1H), 8.33 (s, 1H), 6.94 (m, 1H), 6.68 (b,2H), 6.62 (s, 1H), 6.56 (m, 1H) (2)

225.0/225.2 (d₆-DMSO) 8.97 (s, 1H), 7.45 (m, 1H), 7.19 (m, 1H), 7.09 (d,1H), 6.99 (b, 2H), 3.82 (s, 3H) (3)

246.1/246.3 (d₆-DMSO) 8.85 (s, 1H), 7.18 (m, 2H), 6.92 (d, 2H), 6.83 (m,2H), 3.73 (dd, 4H), 3.07 (dd, 4H) (4)

251.1/251.2 (d₆-DMSO) 8.99 (s, 1H), 6.95 (b, 2H), 6.65 (s, 2H), 3.73 (s,6H), 3.62 (s, 3H) (5)

229.1/229.2 (d₆-DMSO) 9.37 (s, 1H), 7.80 (s, 1H), 7.62 (d, 1H), 7.53 (t,1H), 7.44 (d, 1H) (6)

189.1/189.2 (CDCl₃) 7.19 (s, 1H), 7.09 (m, 3H), 5.46 (b, 2H), 2.38 (s,3H), 2.26 (s, 3H) (7)

189.1/189.2 (CDCl₃) 7.53 (s, 1H), 6.94 (s, 1H), 6.87 (s, 2H), 5.68 (b,2H), 2.32 (s, 6H) (8)

209.1/209.2 (d₆-DMSO) 8.54 (s, 1H), 7.31 (m, 2H), 7.22 (m, 1H), 7.02 (b,2H), 2.17 (s, 3H) (9)

289.0/289.2 (d₆-DMSO) 9.22 (s, 1H), 8.18 (d, 1H), 7.98 (d, 1H), 7.88 (d,1H), 7.72 (m, 2H), 7.46 (m, 1H), 7.14 (b, 2H)

Example 2 REPRESENTATIVE LARGE-SCALE SYNTHESIS OF COMPOUND (1)

[0139] N-Cyano-S-methylisothiourea (576 mg, 5.00 mmol) was dissolved ini-PrOH (7.5 mL). To this stirred solution was added aqueous NaOH (2.0 M,2.5 mL), and the resulting reaction mixture was heated to 100° C. for 30min. The solution was allowed to cool down to ambient temperature. Tothis dicyanamide solution was added a solution of4-hydroxy-2-methylaniline (616 mg, 5.00 mmol) in HCl_((aq)) (1.0 M, 5.0mL). The reaction mixture was heated at 100° C. for 60 min, and aftercooling to ambient temperature, evaporated to dryness. To the crude wasadded an equivalent weight of silica gel and MeOH (10 mL/g crude). Afterstirring for a few minutes, the MeOH was removed by rotary evaporation,and the silica/crude further freed of MeOH by evaporation of addeddichloromethane (DCM, 10 mL/g crude). This coevaporation step wasrepeated three times. The silica/crude mix was placed on top of aflash-SGC equilibrated with DCM/MeOH (95:5). Elution with a stepwisegradient of MeOH in DCM (5-10%) afforded Compound (1) as a light brownsolid after drying under high vacuum. Yield: 482 mg (50.7%).

Example 3 CHONDROCYTE ACTIVITY ASSAY

[0140] Immortalized TC28 (a.k.a. “T/C-28”) juvenile rib chondrocyteswere provided by Dr. Mary Goldring (Harvard Medical School, Boston,Mass.). The TC28 cells were maintained in monolayer culture inDMEM/Ham's F12 (1:1) and supplemented with 10% FCS, 1% L-glutamine, 100units/ml Penicillin and 50 mg/ml Streptomycin (Omega Scientific,Tarzana, Ca) and cultured at 37° C. with 5% CO₂. Additionally, tofurther study chondrocytic cells in a more physiologic nonadherentstate, in some experiments, TC28 cells were transferred to 6 well platesthat had been previously coated for 18 hours at 22° C. with 10% (v/v) in95% ethanol solution of the cell adhesion inhibitor poly 2-hydroxyethylmethacrylate (polyHEME), followed by two washes in PBS. CompleteDMEM/Ham's F12 medium was then added to the wells and the cells studiedfor up to 72 hours in culture (Folkman J. and Moscona A: Role of cellshape in growth control, Nature 273:345-349, 1978; Reginato A, lozzo R,Jimenez S: Formation of Nodular Structures Resembling Mature ArticularCartilage in Long-Term Primary Cultures of Human Fetal EpiphysealChondrocytes on a Hydrogel Substrate, Arthritis Rheum 37: 1338-1349,1994). Type II collagen and aggrecan expression were confirmed usingRT-PCR, which verified maintenance of chondrocyte phenotype.

[0141] The chondrocyte protective effects of representative compoundswere screened in vitro. The agonists included a donor of nitric oxide(NOC-12), a donor of peroxynitrite (SIN-1), and human recombinant IL-1beta. SIN-1 at 100 μ1M and NOC-12 at 250 μM were used as the toxicstimuli for adherent cells. In experiments using TC28 cells cultured inpolyHEME plates, SIN-1 at 10 μM., NOC 12 at 25 μM and IL-1 at 10 ng/mlwere used as the pro-osteorthritic triggers in the absence or presenceof 1 μM of representative compound from Example 1. Cytotoxicity wasstudied using standard LDH release assay, and chondrocyte intracellularATP was measured by standard luciferase assay.

[0142] The enhanced release from chondrocytes of glycosaminoglycans(GAG) is a central feature of osteoarthritic chondrocytes, and is knownto be stimulated potently by IL-1, which, like NO and peroxynitrite is amajor pathogenic factor in osteoarthritis. Thus, GAG release was alsostudied, in which, to optimize the screening assay, a one hour digestionof the cartilage “nodules” formed in the polyheme system was carried outusing 300 μg/ml of papain in 20 mM sodium phosphate, 1 mM EDTA, and 2 mMDTT (pH 6.8). The digestion of the interfering proteins accomplished inthis manner allowed the GAG release to be more readily detectable, andthe GAG release was quantified by the standard dimethylene blue (DMB)dye binding colorimetric assay. In brief, the cell extract digested fromabove was combined with 46 μM DMB, 40 mM glycine and 40 mM NaCl (pH 3.0)and immediately read at 525 nm and compared again at a standard curvegenerated with samples of 1-50 μg/ml chondroitin sulfate (Farndale R,Buttle D, Barrett A: Improved quantitation and discrimination ofsulphated glycosaminoglycans by us of dimethylmethylene blue, Biochimicaet Biophysica Acta 883: 173-177, 1986; SztrolovicsR, White R, Poole R,Mort J, Roughley P: Resistance of small leucine-rich repeatproteoglycans to proteolytic degradation during interleukin-1 stimulatedcartilage catabolism, Biochem J. 339: 571-577, 1999). The results ofthis experiment are presented in Table 2. TABLE 2 % Decrease in GAGRelease Compound NOC-12 IL-1b SIN-1 (1) 27.4 36.8 47.7 (2) 50.1 40.923.6 (3) 28.0 29.1 21.9 (4) 17.2 1.5 11.3 (5) 14.2 30.7 35.0 (6) 11.02.4 1.6 (7) 46.2 56.6 42.3 (8) 32.3 43.8 55.2

Example 4 FURTHER ASSAYS UTILIZING COMPOUND (1)

[0143] Cell Viability Assay

[0144] 1×10⁵ TC28 cells (DMEM/F12 media with 10% FCS, 1% glutamine, 1%P/S) were plated each well in a 96 well plate and allowed to adhereovernight. The cells were washed once with PBS and media changed tocontain only 1% FCS. Compound (1) at various concentrations was added tothe cells for a pretreatment of 1 hr. The media was removed and freshcompound +/− the toxic stimuli were added. The cells were then incubatedfor 24 hrs at 37° C. Following the incubation the media was collectedand used for analysis in the CytTox 96 Nonradioactive Cytotoxicity Assay(Promega, Madison, Wis.). Briefly the LDH release from the dead cellswas quantified in a 30 min enzymatic reaction that results in theconversion of a tetrazolium saletin to a red formazan product. Theresults were then expressed as the percent of cells dead relative to therelease of LDH by the control cells.

[0145] ATP Assay

[0146] 5×10⁵ TC28 cells were plated in a 60 mm dish and allowed toadhere overnight. The cells were then washed with PBS and media waschanged to 1% FCS containing media. Compound (1) was added for 1 hrpretreatment of the cells at 37° C. The media was removed and freshcompound +/− the toxic stimuli were added and the cells incubated for 24hrs at 37° C. The cells were gently scraped into PBS and washed and thenthe pellets were snap frozen in dry ice. The cells were then extractedin 0.4 N perchloric acid and incubated on ice for 15 min. The cells werecentrifuged at 14,000 rpm for 15 min and the supernatant removed. 24%(by volume) of 2.2 M KHCO₃ was added to neutralize the solution and theprecipitate was pelleted by centrifugation. This supernatant was mixedwith the ATP assay mix from the Sigma ATP Luciferase kit and thereaction was counted for 15 sec (with an initial 5 sec delay) intriplicate. Counts were corrected for total DNA in the cell pellet (seeTable 3).

[0147] The results of the above Cell Viability and ATP Assays arepresented in Table 3, which provides the approximate EC₅₀ values (μM)for prevention of SIN-1 and NOC-12 mediated cell death and ATP depletionby Compound (1). In these assays, TC28 cells were pre-treated for onehour followed by 24-hour exposure to trigger in the presence of Compound(1). TABLE 3 Preservation of Cell Viability and ATP Levels in Presenceof Pro-Osteoarthritic Triggers Cell Viability ATP Depletion TriggerNoc12^(a) Sin-1^(b) Noc12^(a) Sin-1^(b) EC₅₀ (μM) 0.01 0.01 0.1 0.1

[0148] Collagen Synthesis

[0149] Collagen production was measured by following ³H prolineincorporation as described in Johnson et al., Arthritis Rheum.43:11560-70, 2000. The results are presented in Table 4, which providesthe approximate EC₅₀ values (μM) for prevention of SIN-1, NOC-12 andIL-1 mediated GAG release (via Example 3) and inhibition of collagensynthesis (via Johnson et al.) in chondrocytes by Compound (1). In theseexperiments, TC28 cells in polyHEME coated plates were pre-treated for 1hr followed by exposure to trigger for 72 hrs in the present of Compound(1).

[0150] In addition, rates of oxygen consumption by TC28 cells inmonolayer culture were also evaluated by the procedures of Johnson etal., the results of which are presented in FIG. 1, which shows thatCompound (1) blocked SIN-1-mediated inhibition of mitochondrialrespiration in TC28 cells. In this experiment, TC28 cells were treatedwith 500 μM SIN-1 for 4 hr +/−10 μM Compound (1): State 3/4—basalrespiration rate with no substrate addition; State 4—respiration rate inpresent of 5 μM/ml oligomycin; State 3U—maximal state 3 uncoupledrespiration rate due to addition of the uncoupler CCCP. TABLE 4 MatrixPreservation in Chondrocytic Cells in Presence of Pro-OsteoarthriticTriggers Collagen GAG Release Trigger Noc12^(a) Sin-1^(b) IL-1^(c)Noc12^(a) Sin-1^(b) IL-1^(c) EC₅₀ (μM) 1 1 1 0.1 0.1 0.1

[0151] Bovine Cartilage Organ Culture Methods

[0152] Mature bovine knees were obtained and cartilage from the femoralcondyles and patellar groove was removed in full thickness slices. (1-3mm). Circular cores (6-7 mm in diameter) were punched out of the tissue.The cores were washed twice with media (1% FCS, 1% P/S, 1% glutaminecontaining DMEM high glucose) and then placed in 96 wells plates. Theslices were incubated in media (as above) at 37° C. for 48 hrs to allowfor recovery from the isolation process. After the recovery period, themedia was removed and fresh media with Compound (1) was added to theslices, for a pretreatment period of 6 hrs. Then the media was removedand fresh Compound (1) +/−IL-1 (at 10 ng/ml) was added and incubated at37° C. for 24 hrs. The conditioned media was collected and the GAG andNO release were analyzed. Finally the slices were weighed to correct forslight variations in size or thickness. The results of this experimentare presented in Table 5, which provides the approximate EC₅₀ values(μM) for prevention of IL-1-mediated GAG and NO release in bovinecartilage slices by Compound (1). TABLE 5 Prevention of MatrixDegradation and Inhibition of NO Release in Bovine Cartilage Slices inResponse to IL-1 Stimulus IL-1 Trigger GAG Release NO Release EC₅₀ M 1010

[0153] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

What is claimed is:
 1. A compound having the structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein R₁, R₂, R₃, R₄ and R₅ are the same or different and individuallyhydrogen, halogen, hydroxy, alkyl, alkoxy, substituted alkyl, aryl,substituted aryl, arylalky, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl or substitutedheterocyclealkyl; or R₃ taken together with R₄, or R₄ taken togetherwith R₅, and further taken together with the respective carbon atom towhich these groups are attached, form an unsubstituted or substitutedfused aryl or heterocycle.
 2. The compound of claim 1 wherein R₁ ishydrogen or alkyl.
 3. The compound of claim 2 wherein R₁ is methyl. 4.The compound of claim 1 wherein R₂ is hydrogen, halogen, alkoxy, alkylor substituted alkyl
 5. The compound of claim 1 wherein R₂ is hydrogen.6. The compound of claim 1 where R₃ is hydrogen, halogen, hydroxy,alkoxy or alkyl.
 7. The compound of claim 1 where R₃ is heterocycle. 8.The compound of claim 7 where R₃ is morpholinyl.
 9. The compound ofclaim 1 wherein R₄ is hydrogen or alkoxy.
 10. The compound of claim 1wherein R₅ is hydrogen.
 11. The compound of claim 1 wherein R₄ takentogether with R₅, and further taken together with the respective carbonatom to which these groups are attached, form an unsubstituted fusedphenyl group.
 12. The compound of claim 1 wherein R₂, R₄ and R₅ arehydrogen.
 13. The compound of claim 12 wherein R₁ is methyl and R₃ ishydroxy.
 14. A pharmaceutical composition comprising a compound of claim1 and a pharmaceutically acceptable carrier.
 15. A method for treatingan arthritic disorder, comprising administering an effective amount of apharmaceutical composition of claim 14 to an animal in need thereof. 16.A method for treating a disease associated with altered mitochondrialfunction comprising administering an effective amount of apharmaceutical composition of claim 14 to an animal in need thereof.